Control of development of biofilms in industrial process water

ABSTRACT

There is provided a method of inhibiting the development of a biofilm adjacent a surface, the method comprising intermittently applying a biofilm inhibiting substance to a collection of microorganisms having biofilm developing potential. Other embodiments are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/211,965, filed Aug. 2, 2002, which claims the benefit of U.S.Provisional Patent Application No. 60/310,623, filed Aug. 6, 2001.

FIELD OF THE INVENTION

The present invention relates to the control of development of biofilmsin industrial process water and water supply lines.

BACKGROUND

Industrial water-carrying vessels, such as process chests, pipes,process water storage tanks, additive tanks, filters, water supply pipesor waste-water pipes, etc., are often observed to have a growth coatingone or more surfaces of the water-carrying vessel where the surfacescontact the water. This growth is actually a biofilm, a collection ofmicroorganisms embedded in a matrix of extracellular polymericsubstances and various organic and inorganic compounds. In the lastseveral years, the nature of these biofilms has been the focus ofattention among both academic and industrial researchers.

Although biofilms may contain a single species of microorganism,typically biofilms comprise not only different species of microorganismsbut different types of microorganisms, for example algae, protozoa,bacteria and others. It has been found that one of the characterizingfeatures of biofilms is that the microorganisms therein actcooperatively or synergistically. Thus, for example, the activity ofcertain enzymes produced by bacteria which are attached to a surface isobserved to be much higher than the corresponding activity of the sameenzymes produced by these bacteria in planktonic form, i.e. whenfree-floating (David G. Davies, in “Microbial Extracellular PolymericSubstances”, Springer-Verlag 1999; Editors: J. Wingender, T. R. New, H.C. Flemming, hereinafter “Wingender et al.”). Comparative studies ofenzyme activities in planktonic bacteria and bacteria attached to solidsurfaces which contact water have shown that enzymatic activity inattached bacteria is greater than in planktonic bacteria (M. Hoffman andAlan W. Decho in Wingender et al.). Communication within microbialbiofilms is responsible for the induction and regulation of theactivities of the biofilm, including for example extracellular enzymebiosynthesis, biofilm development, antibiotic biosynthesis,biosurfactant production, exo-polysaccharide synthesis and more, all ofwhich involve complex biochemical activity (Alan W. Decho in Wingenderet al.). Exchange of genetic material between the microorganisms inbiofilms has also been observed. Empirically it has been found that, ina given industrial water environment, microorganisms living in a biofilmare better protected from biocides than microorganisms living outside abiofilm. Thus, collectively the microorganisms embedded in a biofilmdisplay characteristics which are different from the characteristicswhich are displayed by a like number of planktonic microorganisms.

By acting cooperatively, a collection of microorganisms acts as amicrobial community: it is able to construct a matrix formed ofinorganic and organic material and thus to form and maintain a biofilm.Since microorganisms are single-celled organisms that grow and multiply,the microorganisms in a biofilm must continually replenish the matrixaround them, expand the matrix and maintain the matrix. This process canbe likened to a group of people who act together to construct acontiguous set of dwelling units for themselves, and who then not onlymaintain the existing homes but also add additional homes to accommodatepopulation growth, either by building contiguously horizontally or byadding new homes vertically on top of existing homes.

As scientists best understand it at present, the cooperative behaviorbetween the microorganisms in biofilms is induced by communicationbetween the microorganisms. For example, homoserine lactones play animportant role in communication between bacteria. The extracellularpolymer matrix of a biofilm seems to present an efficient medium forchemical communication and thus to promote more efficient communicationbetween individual microorgansims embedded in the biofilm.

Because microorganisms in biofilms are more effective than planktonicmicroorganisms in producing enzymes, much interest has been shown indeveloping biofilms for effecting chemical reactions. However, in thecontext of industrial and process water-carrying vessels, such asconduits, water tanks and the like, this propensity to produce enzymes,and more importantly the tendency of biofilms to form heavy biomass onthe surface of the vessel, can be extremely detrimental. As a biofilmgrows, it may reduce the effective diameter of a pipe or other conduitat a particular point along the path of the water or increase frictionalong the flow path in the conduit, thus increasing resistance to theflow of water through the conduit, reducing the flow of watertherethrough, increasing power consumption in the pumps which push orpull the water through the conduit, and decreasing the efficiency ofindustrial operations.

Biofilms also deteriorate the quality of various chemicals and processadditives. For example, in the paper industry, biofilms causedeterioration of chemicals like starch and calcium carbonate slurrieswhich are added to the pulp slurries in the wet end processes (K.Jokinen in “Papermaking Chemistry”, Part 4, 1999, Ed. Fapet Oy).Microorganisms are also responsible for hydrogen peroxide degradation inbleaching and de-inking systems (J. F. Kramer, MP Chemical Treatment,August 1997, pp. 42-50). The presence of H₂O₂-degrading enzymes inde-inking and bleaching mills thus necessitates the feeding of largerquantities of hydrogen peroxide than would otherwise be necessary inorder to meet the set point bleaching criteria, thereby increasingproduction costs.

Biofilms may also cause severe corrosion of pipes and chests, may causesevere problems in paper and board machines, and inter alia may causedeterioration of the quality of finished paper, foul odors and severerunnability problems.

Various methods have been described in the prior art in order to controlbiofilms in industry. One approach has been to physically destroy thebiofilm by mechanical means, e.g. by scraping or by sonication. Forexample, U.S. Pat. No. 4,419,248 to Costerson describes a method forremoving biofilm from a surface submerged in water. The method includescooling the surface to below the freezing point of water to therebygenerate large, sharp-edged ice crystals in the biofilm. The frozenbiofilm is then thawed and removed from the surface by, for instance,flowing a liquid across the surface. This approach is often impractical,however, since the place where the biofilm grows may be inaccessible,and/or disruption of industrial operations may be required in order toreach the biofilm.

Another approach has been to physically destroy the biofilm by chemicalmeans, e.g. by use of surface-active agents and detergents which causethe biofilm matrix to break apart. For example, U.S. Pat. No. 5,753,180to Burger describes a non-biocidal method for inhibiting microbiallyinfluenced corrosion susceptible metal surfaces having an anaerobicbiofilm containing active sulfate-reducing bacteria, comprisingcontacting the biofilm with a liquid dispersion of an anthraquinonecompound. U.S. Pat. No. 6,149,822 to Fabri describes a process for bothremoving and controlling biofilms present in industrial cooling andprocess waters. The process provides a composition which includes thereaction products of an amino base, formaldehyde, an alkylenepolyamine,and the ammonium salt of an inorganic or organic acid. The compositionmay be used to remove existing biofilms from process water equipment.Further lower maintenance dosages may be used to maintain the equipmentin a substantially biofilm free condition. U.S. Pat. No. 5,670,055 to Yuet al. describes a method for dispersing biofilms in industrial processwater, which comprises adding an effective biofilm dispersing amount oflinear alkylbenzene sulfonate to industrial process water which containsslime-forming bacteria and other microorganisms. An alternativeembodiment of the invention of Yu et al. comprises adding a compoundselected from the group of biocides cited therein, combined with abiofilm dispersing agent from a list cited therein as well. U.S. Pat.No. 5,382,916 to Wiersma describes a decontamination process forreducing the surface tension of a biofilm, allowing for the removal ofbiofilm and the control of underlying bacteria. In accordance with theinvention of Wiersma, a solution consisting of saponin and soft acidsuch as food grade sodium lactate is contacted with the biofilm. Thesaponin acts as a foaming agent, providing surface tension reductioncapable of loosening the biofilm.

Approaches are known in the art in which the biofilm matrix is degradedby enzymes which are fed externally. For example, U.S. Pat. No.6,100,080 to Johansen describes a method for cleaning and disinfecting asurface at least partly covered by a biofilm layer, comprising the stepsof contacting the biofilm with a cleaning composition comprising one ormore hydrolases, for either fully or partly removing or releasing thebiofilm layer from the surface; and contacting the biofilm with abactericidal disinfecting composition which comprises an oxidoreductasein an amount effective for killing the living bacterial cells present inthe biofilm. Attack with external enzymes leads to loss of activity andchanges in the properties of the biofilm. Such approaches preclude theability of the microorganisms to maintain or expand the matrix. However,such approaches suffer from various drawbacks, for example the treatmentmay be too specific and results may vary in different sites, or thetreatment may not be cost-effective.

An additional difficulty encountered in controlling biofilms inaccordance with the prior art is that as the biofilm matrix decomposes,viable cells are usually released into the water. Such viable cells maystart a new biofilm. Similarly, decomposition of the biofilm matrix maylead to release of enzymes into the water, which may affect theindustrial processes being carried out.

In this regard, biocides can be useful. The use of biocides to treatplanktonic bacteria in industrial process waters is known in the art.See, for example, the inventor's own U.S. Pat. Nos. 5,976,386 and6,132,628, the contents of which are incorporated herein by reference,or U.S. Pat. No. 5,882,526 to Brown et al., which describes a method fortreating regulated waters using a combination of a halogen-containingoxidizer, an erosion control agent, hydrogen peroxide, and a hydrogenperoxide stabilizer. More recently, biocides have been used in attemptsto control biofilms. This goal has sometimes been achieved by combininga biofilm-degrading technique, such as feeding of biofilm-degradingenzymes or physical removal of biofilms, with the application of abiocide which enables the maintenance of a low count of planktonicmicroorganisms in the process water. For example, U.S. Pat. No.5,789,239 to Eyers et. al. describes the use of (a) at least one enzymefrom a defined group to degrade the biofilm and (b) a short-chain glycolas a biocide for the avoidance and/or removal of biofilm on surfaces.U.S. Pat. No. 4,966,716 to Favstritsky et al. describes a method forcontrolling the growth of microorganisms which reduce the efficiency ofrecirculating water systems comprising introducing into such systems abiocidally effective amount of a water soluble perhalide. The perhalideis first introduced in amounts sufficient to kill the microorganisms atfilm forming surfaces of the system. Thereafter, the concentration oforganic ammonium perhalide is maintained at a level sufficient to reducesubstantially the regrowth of such microorganisms.

Alternatively, biocides have been used to control microorganismsembedded in biofilms, i.e. to eradicate the microorganisms themselveswithin the biofilm matrix. Specifically, monochloroamines (MCAs) andfree chlorine (FC) were claimed to show similar efficacy in disinfectingbiofilm bacteria (M. W. LeChevallier et al., Applied and EnvironmentalMicrobiology, pp. 2492-2499, 1988; T. S. Rao et al., Biofouling 12(4)pp. 321-332, 1998). The difficulty with this approach, as stated above,is that empirically it has been found that eradicating microorganisms inbiofilms requires concentrations of biocides which are several timeshigher than the concentrations of biocides required to eradicateplanktonic microorganisms, that long contact times between the biofilmmicroorganisms and the biocide are required, or that continuousapplication of the biocides is required. This increases the cost oftreatment, and may expose workers to greater risks from the biocidesthan is desirable or allowable. It also poses a greater risk to theenvironment.

Approaches to biofilm control utilizing combinations of the abovemethods are also known in the art. These combination approaches, whichare designed in an attempt to solve problems which emerge during theimplementation of each approach separately, may also suffer from some ofthe drawbacks described above. For example, U.S. Pat. No. 6,106,854 toBelfer describes an aseptic disinfectant composition in liquid formhaving germicidal and biofilm cleaning properties comprising ananti-infective, an antiseptic agent, and an anti-biofilm agent forkilling organisms, a water purifying agent for acting as a detergent, asanitizer and a bactericide, a cleansing agent for acting as anastringent and an abradant in the removal of biofilm from contaminatedsurfaces and as a bactericide and fungicide, an anti-oxidant andstabilizer agent, a scrubbing agent for acting as an abrasive and acleanser for the removal of biofilm from contaminated surfaces, at leastone pH adjuster for acidifying the disinfectant composition, and adiluent in the range of 35.0% to 50:0% by weight of the disinfectantcomposition. Barbeau et al., in PCT Patent Publication No. WO 00/27438,describe a composition for removing biofilm. This composition minimallycomprises a detergent, a salt or a salt forming acid, and a bactericide.

A method and composition for suppressing or inhibiting the decompositionaction of enzymes on hydrogen peroxide during bleaching of cellulosefibers with hydrogen peroxide in a way that microorganisms are notmarkedly affected is described in U.S. Pat. No. 5,885,412 to Paart etal. The composition contains hydroxylamine, thiocyanate salts, formicacid, ascorbic acid, or nitrites. It is suggested that the use of one ormore of these substances suppresses or inhibits enzymes such asperoxidases and catalases from decomposing hydrogen peroxide, but doesnot affect microorganisms.

A more recent method for preventing biofilm growth has been to interferewith and prevent the chemical communication between cells in thebiofilm, for example by utilizing antagonists of homoserine lactones. Asin the Biblical story of the Tower of Babel, such approaches directlydisrupt communication between the microorganisms contained in thebiofilm, thus impeding the microorganisms' ability to coordinate theiractions in order to replenish, expand and maintain the matrix, andultimately leading to decomposition of the matrix. For example, Rycroftet al. in PCT Patent Publication no. WO 99/27786 describe compoundswhich may be used in the treatment or prevention of a bacterialinfection in humans or in animals by controlling colonization ofbacteria. The compounds may be employed to remove biofilms fromsurfaces. Davies et al. in PCT Patent Publication No. WO 98/58075describe a method to control the formation, persistence and dispersionof microbial biofilms by taking advantage of the natural process ofcell-cell communication in bacteria. As with treatment by extracellularenzymes, treatment of biofilms in industrial water using antagonists ofhomoserine lactones may be too specific, may yield varying results indifferent sites, or may not be cost-effective.

The present invention seeks to provide a method for controlling thedevelopment of biofilms. The present invention is based on thesurprising observation that the biocides of the inventor's own U.S. Pat.Nos. 5,976,386 and 6,132,628, the contents of both of which areincorporated herein by reference, unexpectedly control biofilmdevelopment, at a feed rate and according to a feeding regime which areinsufficient to cause significant killing of microorganisms embedded inthe biofilms. The unexpectedly low feed rate and feed regime may be usedto maintain biofilm-free surfaces, to remove existing biofilms and tolimit the production of enzymes, including peroxide degrading enzymessuch as catalases, peroxidases and dehydrogenases and starch-degradingenzymes such as amylases, which may otherwise be formed by themicroorganisms embedded in biofilms. Furthermore, the present inventionenables industrial operations involving process waters, such as paperbleaching or de-inking plants, to operate more efficiently, for exampleby reducing the amount of peroxide required during bleaching orde-inking, by reducing the frequency of boil-out, i.e. cleaning thepapermaking machinery with hot, caustic water, and by reducing down-timedue to boil-out and other cleaning operations. The present inventionalso enables optimization of industrial processes which utilize water,including the wet-end chemistry of industrial paper-making processes, bycontrolling the development of biofilms on the surfaces of fibers,suspended particles and additives. It has been recognized by the presentinventor that the growth of biofilms on the surface of fibers andsuspended particles can interfere with the binding of such fibers orparticles, resulting in defects or reduced quality in the resultingpaper.

SUMMARY OF THE INVENTION

There is thus provided in accordance with a preferred embodiment of theinvention a method of inhibiting the development of a biofilm adjacent asurface, the method comprising intermittently applying a biofilminhibiting substance to a collection of microorganisms having biofilmdeveloping potential.

In a preferred embodiment of the invention, said intermittently applyingincludes intermittently administering the biofilm inhibiting substanceto water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, said intermittently applyingcomprises: administering a first discrete amount of a biofilm inhibitingsubstance to water communicating with the collection of microorganisms;waiting for a specified period of time; and thereafter, administering asecond discrete amount of a biofilm inhibiting substance to watercommunicating with the collection of microorganisms.

In a preferred embodiment of the invention, said intermittently applyingcomprises administering a first discrete amount of a biofilm inhibitingsubstance to water communicating with the collection of microorganisms,whereby to obtain a first concentration of the biofilm inhibitingsubstance in the water communicating with the collection ofmicroorganisms allowing the concentration of the biofilm inhibitingsubstance in the water communicating with the collection ofmicroorganisms to fall below said first concentration; and thereafter,administering a second discrete amount of a biofilm inhibiting substanceto water communicating with the collection of microorganisms.

In one preferred embodiment of the invention, the biofilm inhibitingsubstance is applied to the collection of microorganisms periodicallywith a duty cycle of less than 1:2. In another preferred embodiment ofthe invention, the biofilm inhibiting substance is applied to thecollection of microorganisms periodically with a duty cycle of betweenabout 1:5 and 1:10. In another preferred embodiment of the invention,the biofilm inhibiting substance is applied to the collection ofmicroorganisms periodically with a duty cycle of less than 1:10. Inanother preferred embodiment of the invention, the biofilm inhibitingsubstance is applied to the collection of microorganisms periodicallywith a duty cycle of less than 1:25. In a preferred embodiment of theinvention, the biofilm inhibiting substance is applied to the collectionof microorganisms periodically with a duty cycle of less than 1:50.

In a preferred embodiment of the invention, said intermittently applyingcomprises intermittently administering the biofilm inhibiting substancefor a period of between about 5 minutes and about 4 hours at eachintermittent application.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface and each intermittentapplication of the biofilm inhibiting substance is for a period of about3 hours. In another preferred embodiment of the invention, collection ofmicroorganisms is attached to a consumable surface and each intermittentapplication of the biofilm inhibiting substance is for a period of about5 minutes.

In one preferred embodiment of the invention, the biofilm thedevelopment of which is inhibited is adjacent a durable surface. Inanother preferred embodiment of the invention, the biofilm thedevelopment of which is inhibited is adjacent a consumable surface.

In a preferred embodiment of the invention, the collection ofmicroorganisms is located at an interface between water and a surface ofa solid in an industrial water environment.

In a preferred embodiment of the invention, said intermittently applyinga biofilm inhibiting substance includes intermittently generating thebiofilm inhibiting substance in real time. In a preferred embodiment ofthe invention, said intermittently applying further includes supplyingthe biofilm inhibiting substance to the collection of microorganisms asthe biofilm inhibiting substance is generated in real time.

In a preferred embodiment of the intention, said intermittentlygenerating the biofilm inhibiting substance in real time includesproducing a predetermined dilution of a hypochlorite oxidant, producinga predetermined dilution of an ammonium salt, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the biofilm inhibiting substance havingan effective amount of reproducibility, stability and efficacy in situin the mixer.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance in real time includesproducing a predetermined dilution of a hypochlorite oxidant, producinga predetermined dilution of an ammonium salt, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the biofilm inhibiting substance havingan effective amount of reproducibility, stability and efficacy in situin the mixer, and said supplying the biofilm inhibiting substance to thecollection of microorganisms as the biofilm inhibiting substance isgenerated in real time comprises continuously injecting the activebiofilm inhibiting substance, as it is produced in situ in the mixer,from the mixer into water communicating with the collection ofmicroorganisms.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance includes continuously andsynchronously injecting a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injecting a quantity of an ammonium salt into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium salt and continuously andsynchronously injecting the first and second streams into a mixeraccording to a predetermined ratio to produce the biofilm inhibitingsubstance in situ in the mixer.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance includes continuously andsynchronously injecting a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injecting a quantity of an ammonium salt into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium salt and continuously andsynchronously injecting the first and second streams into a mixeraccording to a predetermined ratio to produce the biofilm inhibitingsubstance in situ in the mixer, and said supplying the biofilminhibiting substance to the collection of microorganisms as the biofilminhibiting substance is generated in real time comprises continuouslyinjecting the biofilm inhibiting substance, as it is produced in situ inthe mixer from the mixer into water communicating with the collection ofmicroorganisms.

In a preferred embodiment of the invention, the ammonium salt isselected from the group consisting of ammonium bromide and ammoniumchloride.

In a preferred embodiment of the invention, the biofilm inhibitingsubstance includes an effective amount of bromide activated chloramine.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising inhibiting the biofilm developingpotential of a collection of microorganisms without completelyeradicating the collection of microorganisms.

In a preferred embodiment of the invention, said inhibiting the biofilmdeveloping potential of a collection of microorganisms withoutcompletely eradicating the collection of microorganisms comprisesintermittently applying a biofilm inhibiting substance to a collectionof microorganisms having biofilm developing potential.

In a preferred embodiment of the invention, said intermittently applyingincludes intermittently administering the biofilm inhibiting substanceto water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, said intermittently applyingcomprises: administering a first discrete amount of a biofilm inhibitingsubstance to water communicating with the collection of microorganisms;waiting for a specified period of time; and thereafter, administering asecond discrete amount of a biofilm inhibiting substance to watercommunicating with the collection of microorganisms.

In a preferred embodiment of the invention, said intermittently applyingcomprises administering a first discrete amount of a biofilm inhibitingsubstance to water communicating with the collection of microorganisms,whereby to obtain a first concentration of the biofilm inhibitingsubstance in the water communicating with the collection ofmicroorganisms, allowing the concentration of the biofilm inhibitingsubstance in the water communicating with the collection ofmicroorganisms to fall below said first concentration; and thereafter,administering a second discrete amount of a biofilm inhibiting substanceto water communicating with the collection of microorganisms.

In one preferred embodiment of the invention, the biofilm inhibitingsubstance is applied to the collection of microorganisms periodicallywith a duty cycle of less than 1:2. In another preferred embodiment ofthe invention, the biofilm inhibiting substance is applied to thecollection of microorganisms periodically with a duty cycle of betweenabout 1:5 and 1:10. In another preferred embodiment of the invention,the biofilm inhibiting substance is applied to the collection ofmicroorganisms periodically with a duty cycle of less than 1:10. Inanother preferred embodiment of the invention, the biofilm inhibitingsubstance is applied to the collection of microorganisms periodicallywith a duty cycle of less than 1:25. In another preferred embodiment ofthe invention, the biofilm inhibiting substance is applied to thecollection of microorganisms periodically with a duty cycle of less than1:50.

In a preferred embodiment of the invention, said intermittently applyingcomprises intermittently administering the biofilm inhibiting substancefor a period of between about 5 minutes and about 4 hours at eachintermittent application.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface and each intermittentapplication of the biofilm inhibiting substance is for a period of about3 hours. In another preferred embodiment of the invention, thecollection of microorganisms is attached to a consumable surface andeach intermittent application of the biofilm inhibiting substance is fora period of about 5 minutes.

In a preferred embodiment of the invention, the biofilm the developmentof which is inhibited is adjacent a durable surface. In anotherpreferred embodiment of the invention, the biofilm the development ofwhich is inhibited is adjacent a consumable surface.

In a preferred embodiment of the invention, collection of microorganismsis located at an interface between water and a surface of a solid in anindustrial water environment.

In one preferred embodiment of the invention, said intermittentlyapplying a biofilm inhibiting substance includes intermittentlygenerating the biofilm inhibiting substance in real time. In anotherpreferred embodiment of the invention, said intermittently applyingfurther includes supplying the biofilm inhibiting substance to thecollection of microorganisms as the biofilm inhibiting substance isgenerated in real time.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance in real time includesproducing a predetermined dilution of a hypochlorite oxidant, producinga predetermined dilution of an ammonium salt, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the biofilm inhibiting substance havingan effective amount of reproducibility, stability and efficacy in situin the mixer.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance in real time includesproducing a predetermined dilution of a hypochlorite oxidant, producinga predetermined dilution of an ammonium salt, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the biofilm inhibiting substance havingan effective amount of reproducibility, stability and efficacy in situin the mixer, and said supplying the biofilm inhibiting substance to thecollection of microorganisms as the biofilm inhibiting substance isgenerated in real time comprises continuously injecting the activebiofilm inhibiting substance, as it is produced in situ in the mixer,directly from the mixer into water communicating with the collection ofmicroorganisms.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance includes continuously andsynchronously injecting a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injecting a quantity of an ammonium salt into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium salt and continuously andsynchronously injecting the first and second streams into a mixeraccording to a predetermined ratio to produce the biofilm inhibitingsubstance in situ in the mixer.

In a preferred embodiment of the invention, said intermittentlygenerating the biofilm inhibiting substance includes continuously andsynchronously injecting a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injecting a quantity of an ammonium salt into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium salt and continuously andsynchronously injecting the first and second streams into a mixeraccording to a predetermined ratio to produce the biofilm inhibitingsubstance in situ in the mixer, and said supplying the biofilminhibiting substance to the collection of microorganisms as the biofilminhibiting substance is generated in real time comprises continuouslyinjecting the biofilm inhibiting substance, as it is produced in situ inthe mixer directly from the mixer into water communicating with thecollection of microorganisms.

In a preferred embodiment of the invention, the ammonium salt isselected from the group consisting of ammonium bromide and ammoniumchloride.

In a preferred embodiment of the invention, the biofilm inhibitingsubstance includes an effective amount of bromide activated chloramine.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising inhibiting the biofilm developingpotential of a collection of microorganisms without completelyeradicating the collection of microorganisms by intermittently applyinga biofilm inhibiting substance to a collection of microorganisms havingbiofilm developing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a system for inhibiting the development of a biofilm adjacenta surface, the system comprising an intermittent applicator forintermittently applying a biofilm inhibiting substance to a collectionof microorganisms having biofilm developing potential.

In a preferred embodiment of the invention, the intermittent applicatorincludes an administerer which administers the biofilm inhibitingsubstance to water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, the administerer administersa first discrete amount of a biofilm inhibiting substance to watercommunicating with the collection of microorganisms, and, after aspecified period of time, administers a second discrete amount of abiofilm inhibiting substance to water communicating with the collectionof in microorganisms.

In a preferred embodiment of the invention, the administerer administersa first discrete amount of a biofilm inhibiting substance to watercommunicating with the collection of microorganisms, whereby to obtain afirst concentration of the biofilm inhibiting substance in the watercommunicating with the collection of microorganisms; and, after allowingthe concentration of the biofilm inhibiting substance in the watercommunicating with the collection of microorganisms to fall below saidfirst concentration, administers a second discrete amount of a biofilminhibiting substance to water communicating with the collection ofmicroorganisms.

In one preferred embodiment of the invention, the biofilm inhibitingsubstance is applied to the collection of microorganisms periodicallywith a duty cycle of less than 1:2. In another preferred embodiment ofthe invention, the intermittent applicator applies the biofilminhibiting substance to the collection of microorganisms periodicallywith a duty cycle of between about 1:5 and 1:10. In another preferredembodiment of the invention, the intermittent applicator applies thebiofilm inhibiting substance is the collection of microorganismsperiodically with a duty cycle of less than 1:10. In another preferredembodiment of the invention, the intermittent applicator applies thebiofilm inhibiting substance to the collection of microorganismsperiodically with a duty cycle of less than 1:25. In another preferredembodiment of the invention, the intermittent applicator applies thebiofilm inhibiting substance to the collection of microorganismsperiodically with a duty cycle of less than 1:50.

In a preferred embodiment of the invention, the administerer administersthe biofilm inhibiting substance for a period of between about 5 minutesand about 4 hours at each intermittent application.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface and each intermittentapplication of the biofilm inhibiting substance is for a period of about3 hours. In another preferred embodiment of the invention, thecollection of microorganisms is attached to a consumable surface andeach intermittent application of the biofilm inhibiting substance is fora period of about 5 minutes.

In one preferred embodiment of the invention, the biofilm thedevelopment of which is inhibited is adjacent a durable surface. Inanother preferred embodiment of the invention, the biofilm thedevelopment of which is inhibited is adjacent a consumable surface.

In a preferred embodiment of the invention, the collection ofmicroorganisms is located at an interface between water and a surface ofa solid in an industrial water environment.

In a preferred embodiment of the invention, the intermittent applicatorintermittently generates the biofilm inhibiting substance in real time.

In a preferred embodiment of the invention, the intermittent applicatorfurther supplies the biofilm inhibiting substance to the collection ofmicroorganisms as the biofilm inhibiting substance is generated in realtime.

In a preferred embodiment of the invention, the intermittent applicatorfurther comprises a first producer for producing a predetermineddilution of a hypochlorite oxidant, a second producer for producing apredetermined dilution of an ammonium salt, and a controller forsynchronously metering the two dilutions into a mixer to continuouslymix therein according to a predetermined ratio to produce the biofilminhibiting substance having an effective amount of reproducibility,stability and efficacy in situ in the conduit.

In a preferred embodiment of the invention, the applicator furthercomprises an injector for injecting the active biofilm inhibitingsubstance, as it is produced in situ in the mixer, directly from themixer into water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, in each intermittentgeneration of the biofilm inhibiting substance the system continuouslyand synchronously injects a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injects a quantity of an ammonium salt into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium salt and continuously andsynchronously injects the first and second streams into a mixeraccording to a predetermined ratio to produce the biofilm inhibitingsubstance in situ in the mixer.

In a preferred embodiment of the invention, in each intermittentgeneration of the biofilm inhibiting substance the system continuouslyand synchronously injects a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injects a quantity of an ammonium salt into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium salt and continuously andsynchronously injects the first and second streams into a mixeraccording to predetermined ratio to produce the biofilm inhibitingsubstance in situ in the mixer, and in each intermittent application theapplicator continuously injects the biofilm inhibiting substance, as itis produced in situ in the mixer directly from the mixer into watercommunicating with the collection of microorganisms.

In a preferred embodiment of the invention, the ammonium salt isselected from the group consisting of ammonium bromide and ammoniumchloride.

In a preferred embodiment of the invention, the biofilm inhibitingsubstance includes an effective amount of bromide activated chloramine.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilmcomprising applying to a collection of microorganisms attached to asurface in an industrial water environment at an interface between thesurface and water an amount of bromide activated chloramine effective toinhibit the development of a biofilm by the collection of microorganismswithout completely eradicating the collection of microorganisms.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising intermittently applying bromideactivated chloramine to a collection of microorganisms having biofilmdeveloping potential.

In a preferred embodiment of the invention, each intermittentapplication of the bromide activated chloramine includes producing apredetermined dilution of a hypochlorite oxidant, producing apredetermined dilution of ammonium bromide, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the bromide activated chloramine havingan effective amount of reproducibility, stability and efficacy in situin the mixer and continuously injecting the bromide activatedchloramine, as it is produced in situ in the mixer, directly from themixer into water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, the predetermined dilutionof the oxidant is continuously produced immediately before it issynchronously metered into the mixer with the predetermined dilution ofthe ammonium bromide.

In a preferred embodiment of the invention, the predetermined dilutionof the ammonium bromide is continuously produced immediately before itis synchronously metered into the mixer with the predetermined dilutionof the oxidant.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, has a pH of at least 8.5before being introduced into the water communicating with the collectionof microorganisms.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, has a pH of over 9.5before being introduced into the water communicating with the collectionof microorganisms.

In a preferred embodiment of the invention, the water communicating withthe collection of microorganisms has a pH of between about 5 and about10.5 before the bromide activated chloramine is injected into the water.

In a preferred embodiment of the invention, the water communicating withthe collection of microorganisms has a pH of between about 7 and about 9before the bromide activated chloramine is injected into the water.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in site in the conduit, is injected into thewater communicating with the collection of microorganisms to aconcentration of 0.5-300 ppm expressed as chlorine.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the conduit, is injected into thewater communicating with the collection of microorganisms to aconcentration of 3-10 ppm expressed as chlorine.

In a preferred embodiment of the invention, the ammonium bromide has aconcentration of about 0.1 wt.% to about 50 wt. %.

In a preferred embodiment of the invention, the ammonium bromide has aconcentration of about 2.5 wt. % to about 38 wt. %.

In a preferred embodiment of the invention, the predetermined dilutionof ammonium bromide has a concentration of 0.1 wt. % to 6.0 wt. % and isequimolar to the diluted oxidant solution.

In a preferred embodiment of the invention, the oxidant is selected fromthe group consisting of sodium hypochlorite and calcium hypochlorite.

In a preferred embodiment of the invention, the oxidant is a solution ofhypochlorite, and the ammonium bromide is a solution containing anexcess of base corresponding to at least 10% NaOH.

In a preferred embodiment of the invention, a base is synchronouslyadded to the ammonium bromide to stabilize the bromide activatedchloramine.

In a preferred embodiment of the invention, the oxidant has aconcentration of between 0.1 wt. % and 15 wt. % expressed as Cl₂.

In a preferred embodiment of the invention, the oxidant has aconcentration between 5 wt. % and 15 wt. % expressed as Cl₂.

In a preferred embodiment of the invention, after addition of water theoxidant dilution has a concentration of 0.1 wt. % to 2.0 wt. % expressedas Cl₂.

In a preferred embodiment of the invention, said applying an effectiveamount of bromide activated chloramine includes continuously andsynchronously injecting a quantity of hypochlorite into a first streamof water passing through a first conduit to produce therein apredetermined dilution of the hypochlorite, continuously andsynchronously injecting a quantity of ammonium bromide into a secondstream of water passing through a second conduit to produce therein apredetermined dilution of the ammonium bromide, continuously andsynchronously injecting the first and second streams into a mixeraccording to a predetermined ratio to produce the bromide activatedchloramine in situ in the mixer, and continuously injecting the bromideactivated chloramine, as it is produced in situ in the mixer, directfrom the mixer into water communicating with the collection ofmicroorganisms.

In a preferred embodiment of the invention, the hypochlorite iscontinuously injected into the first stream of water by a first dosingpump connected to a reservoir of the oxidant.

In a preferred embodiment of the invention, the ammonium bromide iscontinuously injected into the second stream of water by a second dosingpump connected to a reservoir of the ammonium bromide and synchronouslyoperated with the first dosing pump.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising intermittently inhibiting the biofilmdeveloping potential of a collection of microorganisms withoutcompletely eradicating the collection of microorganisms.

In a preferred embodiment of the invention, said inhibiting the biofilmdeveloping potential of a collection of microorganisms withoutcompletely eradicating the collection of microorganisms comprisesapplying bromide activated chloramine to a collection of microorganismshaving biofilm developing potential.

In a preferred embodiment of the invention, each intermittentapplication of the bromide activated chloramine includes producing apredetermined dilution of a hypochlorite oxidant, producing apredetermined dilution of ammonium bromide, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the bromide activated chloramine havingan effective amount of reproducibility, stability and efficacy in situin the mixer and continuously injecting the bromide activatedchloramine, as it is produced in situ in the mixer, directly from themixer into water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, the predetermined dilutionof the oxidant is continuously produced immediately before it issynchronously metered into the mixer with the predetermined dilution ofthe ammonium bromide.

In a preferred embodiment of the invention, the predetermined dilutionof the ammonium bromide is continuously produced immediately before itis synchronously metered into the mixer with the predetermined dilutionof the oxidant.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, has a pH of at least 8.5before being introduced into the water communicating with the collectionof microorganisms.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, has a pH of over 9.5before being introduced into the water communicating with the collectionof microorganisms.

In a preferred embodiment of the invention, the water communicating withthe collection of microorganisms has a pH of between about 5 and about10.5 before the bromide activated chloramine is injected into the water.

In a preferred embodiment of the invention, the water communicating withthe collection of microorganisms has a pH of between about 7 and about 9before the bromide activated chloramine is injected into the water.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, is injected into the watercommunicating with the collection of microorganisms to a concentrationof 0.5-300 ppm expressed as chlorine.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, is injected into the watercommunicating with the collection of microorganisms to a concentrationof 3-10 ppm expressed as chlorine.

In a preferred embodiment of the invention the ammonium bromide has aconcentration of bout 0.1 wt. % to about 50 wt. %.

In a preferred embodiment of the invention, the ammonium bromide has aconcentration of about 2.5 wt. % to about 38 wt. %.

In a preferred embodiment of the invention, the predetermined dilutionof ammonium bromide has a concentration of 0.1 wt. % to 6.0 wt. % and isequimolar to the diluted oxidant solution.

In a preferred embodiment of the invention, the oxidant is selected fromthe group consisting of sodium hypochlorite and calcium hypochlorite.

In a preferred embodiment of the invention, the oxidant is a solution ofhypochlorite, and the ammonium bromide is a solution containing anexcess of base corresponding to at least 10% NaOH.

In a preferred embodiment of the invention, a base is synchronouslyadded to the ammonium bromide to stabilize the bromide activatedchloramine.

In a preferred embodiment of the invention, the oxidant has aconcentration of between 0.1 wt. % and 15 wt. % expressed as Cl₂.

In a preferred embodiment of the invention, the oxidant has aconcentration between 5 wt. % and 15 wt. % expressed as Cl₂.

In a preferred embodiment of the invention, after addition of water theoxidant dilution has a concentration of 0.1 wt. % to 2.0 wt. % expressedas Cl₂.

In a preferred embodiment of the invention, applying an effective amountof bromide activated chloramine includes continuously and synchronouslyinjecting a quantity of hypochlorite into a first stream of waterpassing through a first conduit to produce therein a predetermineddilution of the hypochlorite, continuously and synchronously injecting,a quantity of ammonium bromide into a second stream of water passingthrough a second conduit to produce therein a predetermined dilution ofthe ammonium bromide, continuously and synchronously injecting the firstand second streams into a mixer according to a predetermined ratio toproduce the bromide activated chloramine in situ in the mixer, andcontinuously injecting the bromide activated chloramine, as it isproduced in situ in the mixer, directly from the mixer into watercommunicating with the collection of microorganisms.

In preferred embodiment or the invention, the hypochlorite iscontinuously injected into the first stream of water by a first dosingpump connected to a reservoir of the hypochlorite.

In a preferred embodiment of the invention, the ammonium bromide iscontinuously injected into the second stream of water by a second dosingpump connected to a reservoir of the ammonium bromide and synchronouslyoperated with the first dosing pump.

There is also provided, in accordance with a preferred embodiment of theinvention, a growth-controlled biomass comprising a collection ofmicroorganisms and bromide activated chloramine at a concentrationeffective to inhibit development of a biofilm by the collection ofmicroorganisms.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

There is also provided, in accordance with a preferred embodiment of theinvention, a growth-controlled biomass comprising a collection ofmicroorganisms and bromide activated chloramine at a concentrationeffective to destroy the biofilm developing potential of the collectionof microorganisms without completely eradicating the collection ofmicroorganisms.

In a preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface.

In a preferred embodiment of the invention, the collection ofmicroorganisms is attached to a consumable surface.

There is also provided, in accordance with a preferred embodiment of theinvention, a growth-controlled biomass including a collection ofmicroorganisms and a biofilm inhibiting substance present at thecollection of microorganisms at a concentration and for a durationeffective to inhibit development of a biofilm by the collection ofmicroorganisms. In one preferred embodiment of the invention, thecollection of microorganisms is attached to a durable surface. Inanother preferred embodiment of the invention, the collection ofmicroorganisms is attached to a consumable surface.

There is also provided, in accordance with a preferred embodiment of theinvention, a growth-controlled biomass including a collection ofmicroorganisms having biofilm developing potential and a biofilminhibiting substance present at the collection of microorganisms at aconcentration and for a duration effective to destroy the biofilmdeveloping potential of the collection of microorganisms withoutcompletely destroying the collection of microorganisms. In one preferredembodiment of the invention, the collection of microorganisms isattached to a durable surface. In another preferred embodiment of theinvention, the collection of microorganisms is attached to a consumablesurface.

There is also provided, in accordance with a preferred embodiment of theinvention, a system for inhibiting the development of a biofilm in anindustrial water environment including a real time bromide activatedchloramine generator adapted to supply bromide activated chloramine inreal time at an interface between water and a surface of a solid in anindustrial water environment.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilm in anindustrial water environment including generating bromide activatedchloramine in real time and applying the bromide activated chloramine inreal time at an interface between water and a surface of a solid in anindustrial water environment.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the production of an enzyme by acollection of microorganism attached to a surface, the method comprisingintermittently applying to a collection of microorganisms attached to asurface a substance which inhibits the production of an enzyme by thecollection of microorganisms.

In a preferred embodiment of the invention, the collection ofmicroorganisms is attached to surface in an industrial waterenvironment.

In one preferred embodiment of the invention, the surface is a durablesurface. In another preferred embodiment of the invention, the surfaceis a consumable surface.

In a preferred embodiment of the invention, the substance does notcompletely eradicate the collection of microorganisms.

In a preferred embodiment of the invention, the substance does notinactivate the enzyme. In a preferred embodiment of the invention, theenzyme is a hydrogen peroxide-degrading enzyme (HPDE), preferably acatalase, a dehyrogenase or a peroxidase. In another preferredembodiment of the invention, the enzyme is a starch-degrading enzyme,preferably an amylase.

In a preferred embodiment of the invention, the collection ofmicroorganisms is present at an interface between water and a surface ofsolid in an industrial environment.

In a preferred embodiment of the invention, the substance is bromideactivated chloramine.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the production of an enzyme by acollection of microorganisms attached to a surface in an industrialwater environment, the method comprising intermittently applying to acollection of microorganisms attached to a surface in an industrialwater environment a substance which inhibits the production of an enzymeby the collection of microorganisms

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the production of an enzyme by acollection of microorganisms adjacent a surface, the method comprisingintermittently applying an enzyme production inhibiting substance to acollection of microorganisms adjacent a surface which have enzymeproducing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the production of an enzyme by acollection of microorganisms adjacent a surface, the method comprisinginhibiting the enzyme producing potential of the collection ofmicroorganisms without complete eradicating the collection ofmicroorganisms.

In a preferred embodiment of the invention, said inhibiting the enzymeproducing potential of the collection of microorganisms withoutcompletely eradicating the collection of microorganisms comprisesintermittently applying an enzyme production inhibiting substance to acollection of microorganisms adjacent a surface which have enzymeproducing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a system for reducing the production of an enzyme by acollection or microorganisms attached to a surface, the systemcomprising an intermittent applicator for intermittently applying anenzyme production inhibiting substance to a collection of microorganismshaving enzyme production potential which are attached to a surface.

In a preferred embodiment of the invention, the intermittent applicatorincludes an administerer which administers the enzyme productioninhibiting substance to water communicating with the collection ofmicroorganisms.

In a preferred embodiment of the invention, the administerer administersa first discrete amount of an enzyme production inhibiting substance towater communicating with the collection of microorganisms and after aspecified period of time administers a second discrete amount of anenzyme production inhibiting substance to the water communicating withthe collection of microorganisms.

In a preferred embodiment of the invention, the administerer administersa first discrete amount of an enzyme production inhibiting substance towater communicating with the collection of microorganisms, whereby toobtain a first concentration of the enzyme production inhibitingsubstance in the water communicating with the collection ofmicroorganisms; and then, after the concentration of the enzymeproduction inhibiting substance in the water communicating with thecollection of microorganisms falls below the first concentration,administers a second discrete amount of an enzyme production inhibitingsubstance to water communicating with the collection of microorganisms.

In a preferred embodiment of the invention, the enzyme productioninhibiting substance does not completely eradicate the collection ofmicroorganisms.

In a preferred embodiment of the invention, the enzyme productioninhibiting substance does not inactivate the enzyme. In one preferredembodiment of the invention, the enzyme is a hydrogen peroxide degradingenzyme, preferably a catalase, a dehydrogenase or a peroxidase. Inanother preferred embodiment of the invention, the enzyme is a starchdegrading enzyme, preferably an amylase.

In a preferred embodiment of the invention, the collection ofmicroorganisms is present at an interface between water and a surface ofa solid in an industrial environment.

In a preferred embodiment of the invention, the enzyme productioninhibiting substance is bromide activated chloramine.

In one preferred embodiment of the invention, the enzyme productioninhibiting substance is presented at the collection of microorganismsperiodically with a duty cycle of less than 1:2. In another preferredembodiment of the invention, the enzyme production inhibiting substanceis presented at the collection of microorganisms periodically with aduty cycle of between about 1:5 and 1:10. In another preferredembodiment of the invention, the enzyme production inhibiting substanceis presented at the collection of microorganisms periodically with aduty cycle of less than 1:10. In another preferred embodiment of theinvention, the enzyme production inhibiting substance is presented atthe collection of microorganisms periodically with a duty cycle of lessthan 1:25. In another preferred embodiment of the invention, the enzymeproduction inhibiting substance is presented at the collection ofmicroorganisms periodically with a duty cycle of less than 1:50.

In a preferred embodiment of the invention, the intermittent applicatorintermittently generates the enzyme production inhibiting substance inreal time.

In a preferred embodiment of the invention, the intermittent applicatorfurther supplies the enzyme production inhibiting substance to thecollection of microorganisms as the enzyme production inhibitingsubstance is generated in real time.

In a preferred embodiment of the invention, the intermittent applicatorincludes a first producer for producing a predetermined dilution ofhypochlorite, a second producer for producing a predetermined dilutionof an ammonium salt, and a controller for continuously and synchronouslymixing the two dilutions in a mixer according to a predetermined ratioto produce the enzyme production inhibiting substance in situ in themixer.

In a preferred embodiment of the invention, the intermittent applicatorincludes an injector for continuously injecting the enzyme productioninhibiting substance, as it is produced in situ in the mixer, directlyfrom the mixer into water communicating with the collection ofmicroorganisms.

In a preferred embodiment of the invention, the ammonium salt isselected from the group consisting of ammonium chloride and ammoniumbromide.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the production of an enzyme by acollection of microorganisms, the method comprising administering to acollection of microorganisms at an interface between water and a surfaceof a solid in an industrial water environment an amount of bromideactivated chloramine effective to inhibit production of an enzyme by thecollection of microorganisms without completely eradicating thecollection of microorganisms.

In a preferred embodiment of the invention, the bromide activatedchloramine does not inactivate the enzyme.

In one preferred embodiment of the invention, the enzyme is a hydrogenperoxide destroying enzyme, preferably a catalase, a dehydrogenase or aperoxidase. In another preferred embodiment of the invention, the enzymeis a starch degrading enzyme, preferably an amylase.

In a preferred embodiment of the invention, said administering bromideactivated chloramine includes producing a predetermined dilution of ahypochlorite oxidant, producing a predetermined dilution of ammoniumbromide, synchronously metering the two dilutions into a mixer tocontinuously mix therein according to a predetermined ratio to producethe bromide activated chloramine having an effective amount ofreproducibility, stability and efficacy in situ in the mixer andcontinuously injecting the bromide activated chloramine, as it isproduced in situ in the mixer, directly from the mixer into watercommunicating with the collection of microorganisms.

In a preferred embodiment of the invention, the predetermined dilutionof the oxidant is continuously produced immediately before it issynchronously metered into the mixer with the predetermined dilution ofthe amine source.

In a preferred embodiment of the invention, the bromide activatedchloramine, as produced in situ in the mixer, has a pH of at least 8.5,preferably over 9.5, before being introduced into water communicatingwith the collection of microorganisms. In a preferred embodiment of theinvention, the water communicating with the collection of microorganismshas a pH of 5-10.5, preferably 7-9, before the bromide activatedchloramine is injected into it.

There is also provided, in accordance with a preferred embodiment of theinvention, an enzyme production-controlled biomass comprising acollection of microorganisms and bromide activated chloramine at aconcentration effective to inhibit production of an enzyme by thecollection of microorganisms.

In one preferred embodiment of the invention the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

There is also provided, in accordance with a preferred embodiment of theinvention, an enzyme production-controlled biomass comprising acollection of microorganisms and bromide activated chloramine at aconcentration effective to destroy the enzyme production potential ofthe collection of microorganisms without completely eradicating thecollection of microorganisms.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

There is also provided, in accordance with a preferred embodiment of theinvention, an enzyme production-controlled biomass including acollection of microorganisms attached to a surface and an enzymeproduction inhibiting substance intermittently present at the collectionof microorganisms at a concentration and for a duration effective toinhibit production of an enzyme by the collection of microorganisms.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

There is also provided, in accordance with a preferred embodiment of theinvention, an enzyme production-controlled biomass including acollection of microorganisms having enzyme production potential and anenzyme production inhibiting substance present at the collection ofmicroorganisms at a concentration and for a duration effective todestroy the enzyme production potential of the collection ofmicroorganisms without completely destroying the collection ofmicroorganisms.

In one preferred embodiment of the invention, the collection ofmicroorganisms is attached to a durable surface. In another preferredembodiment of the invention, the collection of microorganisms isattached to a consumable surface.

In one preferred embodiment of the invention, the enzyme is a hydrogenperoxide-degrading enzyme, preferably a catalase, a dehydrogenase or aperoxidase. In another preferred embodiment of the invention, the enzymeis a starch degrading enzyme, preferably an amylase.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilm adjacenta surface, the method composing intermittently applying a biofilminhibiting substance comprising bromide activated chloramine and aperoxide to a collection of microorganisms having biofilm developingpotential.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilm adjacenta surface, the method comprising inhibiting the biofilm developingpotential of a collection of microorganisms without completelyeradicating the collection of microorganisms by intermittently applyingto the collection of microorganisms a biofilm inhibiting substancecomprising bromide activated chloramine and a peroxide.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for increasing the persistence of hydrogen peroxidein de-inking or bleaching process water, the method comprisingintermittently applying to a collection of microorganisms at aninterface between a surface of a solid and the de-inking or bleachingprocess water a substance that inhibits the production of a hydrogenperoxide-degrading enzyme.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for increasing the persistence of hydrogen peroxidein de-inking or bleaching process water, the method comprisinginhibiting the hydrogen peroxide degrading enzyme production potentialof a collection of microorganisms at an interface between a surface of asolid and the de-inking or bleaching process water by applying to thecollection of microorganisms a substance that inhibits the hydrogenperoxide-degrading enzyme production potential of the collection ofmicroorganisms without completely eradicating the collection ofmicroorganisms.

In a preferred embodiment of the invention, the biofilm inhibitingsubstance includes bromide activated chloramine and the process watercontains peroxide.

In a preferred embodiment of the invention, the biofilm inhibitingsubstance does not degrade peroxide.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for controlling a biofilm, comprising applying to abiofilm locus in need of control an amount of a bromide activatedchloramine efficacious to disrupt the functioning of the biofilm withouteradicating the collection of microorganisms contained in the biofilm.

There is also provided, in accordance with a preferred embodiment of theinvention, an aqueous solution comprising a bromide activated chloramineand a peroxide.

In a preferred embodiment of the invention, the concentration of bromideactivated chloramine is between about 1 part per million (ppm) and about10 ppm, expressed as total chlorine.

In a preferred embodiment of the invention, the concentration ofperoxide is between about 100 ppm and about 40,000 ppm.

In one preferred embodiment of the invention, the solvent of the aqueoussolution is water having a high chlorine demand. In another preferredembodiment of the invention, the solvent of the aqueous solution iswater having a low chlorine demand.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising intermittently applying a biofilminhibiting substance intentionally at a collection of microorganismshaving biofilm developing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising intentionally inhibiting the biofilmdeveloping potential of a collection of microorganisms withoutcompletely eradicating the collection of microorganisms.

In a preferred embodiment of the invention, said intentionallyinhibiting the biofilm developing potential of a collection ofmicroorganisms without completely eradicating the collection ofmicroorganisms comprises intermittently applying a biofilm inhibitingsubstance intentionally to a collection of microorganisms having biofilmdeveloping potential.

There is also provided in accordance with a preferred embodiment of theinvention, a system for inhibiting the development of a biofilm adjacenta surface, the system comprising an intermittent applicator forintermittently applying a biofilm inhibiting substance intentionally toa collection of microorganisms having biofilm developing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilmcomprising intentionally applying to a collection of microorganismsattached to a surface in an industrial water environment at an interfacebetween the surface and water an amount of bromide activated chloramineeffective to inhibit the development of a biofilm by the microorganismswithout completely eradicating the collection of microorganisms.

There is also provided, in accordance with a preferred embodiment of theinvention, a method of inhibiting the development of a biofilm adjacenta surface, the method comprising intermittently applying bromideactivated chloramine intentionally to a collection of microorganismshaving biofilm developing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a growth-controlled biomass including a collection ofmicroorganisms attached to a surface and a biofilm inhibiting substanceintermittently present intentionally at the collection of microorganismsat a concentration and for a duration effective to inhibit developmentof a biofilm by the collection of microorganisms.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the growth of a biofilm in anindustrial water environment including generating bromide activatedchloramine in real time and applying the bromide activated chloramine inreal time at intentionally an interface between water and a surface of asolid in an industrial water environment.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for reducing the production of an enzyme by acollection of microorganisms, the method comprising intermittentlyapplying intentionally to a collection of microorganisms attached to asurface a substance which inhibits the production of an enzyme by thecollection of microorganisms.

There is also provided, in accordance with a preferred embodiment of theinvention, a system for reducing the production of an enzyme by acollection of microorganisms attached to a surface, the systemcomprising an intermittent applicator for intermittently applying anenzyme production inhibiting substance intentionally to a collection ofmicroorganisms having enzyme production potential which are attached toa surface.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the production of an enzyme by acollection of microorganisms, the method comprising intentionallyadministering to a collection of microorganisms at an interface betweenwater and a surface of a solid in an industrial water environment anamount of bromide activated chloramine effective to effect inhibition ofproduction of an enzyme by the collection of microorganisms withoutcompletely eradicating the collection of microorganisms.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilm adjacenta surface, the method comprising applying a biofilm inhibitingcomprising bromide activated chloramine and a peroxide intentionally toa collection of microorganisms having biofilm developing potential.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilm adjacenta surface, the method comprising inhibiting the biofilm developingpotential of a collection of microorganisms without completelyeradicating the collection of microorganisms by intermittently applyingintentionally to the collection of microorganisms a biofilm inhibitingsubstance comprising bromide activated chloramine and a peroxide.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for increasing the persistence of hydrogen peroxidein de-inking or bleaching process water, the method comprisingintermittently applying intentionally to a collection of microorganismsat an interface between a surface of a solid and the de-inking orbleaching process water a substance that inhibits the production of ahydrogen peroxide-degrading enzyme.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for increasing the persistence of hydrogen peroxidein de-inking or bleaching process water, the method comprisinginhibiting the hydrogen peroxide degrading enzyme production potentialof a collection of microorganisms at an interface between a surface of asolid and the de-inking or bleaching process water by applyingintentionally to the collection of microorganisms a substance thatinhibits the hydrogen peroxide-degrading enzyme production potential ofthe collection of microorganisms without completely eradicating thecollection of microorganisms.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for controlling a biofilm, comprising intentionallyapplying, to a biofilm locus in need of control an amount of a bromideactivated chloramine efficacious to disrupt the functioning of thebiofilm without eradicating the collection of microorganisms containedin the biofilm.

There is also provided, in accordance with a preferred embodiment of theinvention, a method for inhibiting the development of a biofilm in anindustrial water environment including generating bromide activatedchloramine in real time and applying the bromide activated chloramineintentionally in real time at an interface between water and a surfaceof a solid in an industrial water environment to inhibit the developmentof a biofilm thereat.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The invention is more particularly described with respect to a number ofexamples set forth below, and also with respect to the accompanyingdrawings wherein:

FIG. 1 is a block diagram illustrating one form of apparatus constructedand operative to enable the practice of the present invention;

FIG. 2 is a similar block diagram illustrating another apparatusconstructed and operative to enable the practice of the presentinvention;

FIG. 3 is a graph of the difference between the Hazen-Williamscoefficient in a pipe treated in accordance the present invention and anuntreated control pipe;

FIG. 4 is a graph comparing differences between pipes treated with abiofilm inhibiting substance, chloramine and an untreated control pipe;

FIG. 5 is a graph showing the incidence in holes and spots in paperfollowing the onset of intermittent application of a biofilm-inhibitingsubstance to a biofilm growing in a paper making machine, in accordancewith the present invention, wherein the machine was not cleaned prior totreatment;

FIG. 6. is a graph showing the incidence of holes and spots in paperfollowing cleaning of the paper making machine and subsequentcontinuation of intermittent application of a biofilm-inhibitingsubstance, in accordance with the present invention;

FIG. 7 is a graph showing the counts of different types of viable cellsin a paper making machine in response to intermittent application of abiofilms inhibiting substance, in accordance with the present invention;and

FIG. 8 is a graph showing the effect of addition of a biofilm inhibitingsubstance on the retention of fibers in a papermaking machine.

The term “duty cycle” will be understood to mean the ratio between (a)the amount of time the biofilm inhibiting substance or enzyme productioninhibiting substance is administered to a collection of microorganismshaving biofilm developing potential and (b) the amount of time such asubstance is not administered to collection of microorganisms havingbiofilm developing potential or enzyme developing potential. In apreferred embodiment of the present invention, the biofilm inhibitingsubstance or the enzyme production inhibiting substance is continuouslyinjected as it is produced into water communicating with a collection ofmicroorganisms having biofilm developing potential. In connection withthis preferred embodiment of the invention, the term “duty cycle” willbe understood to mean the ratio between (a) the amount of time thebiofilm inhibiting substance or enzyme production inhibiting substanceis continuously injected as it is produced into water communicating witha collection of microorganisms having biofilm developing potential orenzyme developing potential and (b) the amount of time such a substanceis not injected into water communicating with a collection ofmicroorganisms having biofilm developing potential or enzyme developingpotential. Thus, if a biofilm inhibiting substance is injected intoprocess water for three hours once in three days in order to inhibitbiofilm development, the duty cycle is 1:23.

In the context of this patent application, the term “excess of basecorresponding to at least 10% NaOH” means a solution containing theequivalent of more than 2 moles of NaOH per mole of Cl₂, calculatedbased on the formation of NaOCl from Cl₂ and NaOH according to theequation:

2NaOH+Cl₂→NaCl+H₂O+NaOCl,

so that the solution contains excess NaOH, and the total amount of NaOH,calculated as the sum of free NaOH and NaOH as represented by NaOCl, isat least 10%.

In the context the present patent application, the term “wet endchemistry” will be understood to be as defined in the IHandbook of Pulpand Paper Terminology by G. A. Smook, Cegep de Trois-Rivieres, 1990.Smook defines wet end chemistry as “Physical and surface chemistry offines and additives and their interaction with fibers.”

In the context of the present patent application, it will be understoodthat the term “a collection of microorganisms attached to a surface”does not imply that each and every microorganism which is part of thecollection is itself necessarily directly attached to the surface. Forexample, a collection of microorganisms which is of several cells'thickness may have a first layer of cells which are attached directly tothe surface, and several additional layers of cells stacked upon thelowest layer. Similarly, microorganisms in a biofilm do not necessarilytouch the surface to which the biofilm is attached, but are embedded inthe matrix of the biofilm. For the purposes of the present patentapplication, such a collection of microorganism is also considered acollection of microorganisms attached to a surface.

The phrase “development of a biofilm” will be understood to encompassboth the creation of a biofilm ab initio by a collection ofmicroorganisms as well as the maintainence or expansion of an existingbiofilm by a collection of microorganisms.

In the context of the present patent application, “durable surface”refers to a surface of an industrial process apparatus, such as thesurface of a pipe, water chest, or other vessel, which is not consumedduring production and which contacts process water. “Consumable surface”refers to a surface, such as the surface of fibers or suspendedparticles present in the process waters, which during the productioncycle may be consumed and exit the apparatus, e.g. as a paper product.

Depending on the type of industrial process, consumable surfaces may bepresent in the apparatus for significantly less time than durablesurfaces, in which case the frequency of treatment or the duty cycle maybe determined by the frequency or duty cycle required to treat thedurable surfaces.

Conversely, in some processes:

-   -   (a) consumable surfaces may be present in the process apparatus        for relatively long periods of time, e.g. in cases where some of        the process water is recycled back into the process stream,    -   (b) the consumable surfaces may be coated with wet-end chemicals        upon which microorganisms can feed,    -   (c) the process water may contain a relatively high        concentration of consumable surfaces (particles and/or fibers),        or    -   (d) the particles or fibers bearing the consumable surfaces are        likely to precipitate.        In such cases the frequency or duty cycle will be determined by        the frequency or duty cycle required to treat the consumable        surfaces.

Particularly with respect to situations (c) and (d), it is noted that inpapermaking, fibers are formed into paper by coating a plastic or wiremesh with a sheet of suspension containing a mixture of fibers, pigmentsand chemicals, as is well-known in the papermaking art, and then througha series of steps the sheet is dried to a water content of about 8%.“Retention” is defined by Smook at p. 191 as the amount of anypapermaking material which is retained in the paper forming process,usually expressed as a percentage of what was initially added. Thus thegreater the percentage of fibers which are retained by the mesh, thehigher the “retention” of the papermaking process. A retention of 90% isconsidered excellent; a retention of 50% is considered poor. Thosefibers which do not become part of the sheet of paper are recycled forfurther use.

In papermaking machines having low or poor retention, the concentrationof fibers in certain parts of the machinery may be higher than inmachines having good retention. Furthermore, because fibers have a largesurface area to mass ratio, and because the fibers used in papermakingare porous, further increasing the surface area to mass ratio, the totalsurface presented by the fibers (which in the context of the presentapplication constitute consumable surfaces) may far exceed the totalsurface presented by the machinery itself. Moreover, because ofrecycling of the fibers, the effective time that some of the fibers arepresent in the papermaking machine may be on the order of hours or evendays.

Consequently, without wishing to be bound by a particular theory, theinventor believes that there exists the opportunity for biofilms to formon the surfaces of fibers from which paper is made, and that thepresence of such biofilms may have a detrimental effect on paperproduction, as the ability of fibers to adhere to one another is crucialfor the formation of paper of acceptable quality, and the presence ofbiofilm on the fibers interferes with such adherence. Poor adherencebetween fibers also increases the likelihood of such fibersprecipitating. Furthermore, it is believed that the problem of biofilmformation on fibers may be exacerbated by the use of certain chemicals,such as starch or sugar in the wet end chemistry of the papermakingprocess, since these chemicals may encourage the growth of biofilms onthe fibers.

The apparatus illustrated in FIG. 1 provides a biofilm inhibitingsubstance to a collection of microorganisms 1 attached to a surfacelocated at a location schematically denoted 2 in the drawing. Thelocation may be, for example, a conduit carrying water or part of apaper making machine, and the surface may be a durable surface or aconsumable surface, as defined hereinabove. The biofilm inhibitingsubstance is applied to the collection of microorganisms 1 byintroducing the biofilm inhibiting substance to a liquid 3, such aswater, which is in communication with the collection of microorganisms1. The biofilm inhibiting substance is formed by mixing in situ twosolutions, namely an oxidant solution, preferably a hypochloritesolution, within a reservoir 4, and an amine source solution, preferablyan ammonium salt solution, within a reservoir 6.

As shown in FIG. 1, water, e.g., tap water is fed from a source 8 via awater pipe 10 through a pair of branch lines 12, 14, connected inparallel to each other, to a mixer 21 which feeds common outlet pipe 16leading to the liquid 3 at the location 2. Each of the two parallelbranch lines 12, 14, includes a venturi tube 18, 20 having an inlet port18 a, 20 a, connected in the respective branch line 12, 14, and anoutlet port 18 b, 20 b, connected to mixer 21 which connects to thecommon outlet line 16 leading to the liquid in communication with thecollection of microorganisms. Each of the venturi tubes 18, 20, includesa third port 18 c, 20 c, leading to the reservoir 4, 6, of therespective solution to be added to the water flowing through the outletline 16.

The two venturi tubes 18, 20, thus constitute dosing pumps whichcontinuously and synchronously inject both oxidant solution fromreservoir 4, and the amine source solution from reservoir 6, into thewater from source 8 in proportions which are predetermined for optimalformation of the biofilm inhibiting substance. These two chemicals aremixed in mixer 21 and react with each other in mixer 21 which feeds intooutlet pipe 16, so that the reaction product, namely the biofilminhibiting substance produced by the reaction of these two chemicals, isintroduced into the liquid 3 as it is produced in situ.

The two branch lines 12, 14 for the two venturi tubes 18, 20 includecontrol valves 22, 74, which enable the flow rate of the water to becontrolled via the two venturi tubes 8, 20. Lines 26, 28 connecting thetwo reservoirs 4,6 to their respective venturi tubes 18, 20 also includevalves, shown at 30, 32, for controlling the dosage of the chemicalsinto the water passing through the venturi tubes. The latter valves alsoenable the supply of chemicals to be terminated at the end of theintroduction of the biofilm inhibiting substance, so that continued flowof the water via the branch lines 12, 14, mixer 21 and the outlet line16 will wash away any residue of these chemicals, or their decompositionproducts, and thereby avoid accumulation of decomposition products whichmay form at the end of each biofilm inhibiting substance productioncycle in the outlet line 16 or in mixer 21.

The control of the foregoing valves is done by a control system,schematically illustrated by block 40. The pH of the biofilm inhibitingsubstance decreases as the biofilm inhibiting substance decomposes.Outlet line 16, therefore, may also and preferably does include a pHsensor 47 for sensing the pH of the biofilm inhibiting substance, andcontrolling the control system 40 in response thereto.

Control system 40 also controls the supply of the water from source 8via an electrical valve 48. Control system 40 can further control analarm 50 or other signalling device. The illustrated system may furtherinclude a timer 52 which is presettable to fix both the lengths of timefor which the biofilm inhibiting substance is to be fed via the outletline 16 to the water communicating with the collection ofmicroorganisms, as well as the time intervals between such feedings ofthe biofilm inhibiting substance.

The water supply line 10 from the water source 8 to the two branch lines12, 14, may include additional control devices. For purposes ofillustration, the accompanying drawings schematically illustrate thefollowing additional control devices: a manual control valve 53,enabling manual control of the water flow from the source 8; a pressurereducer 54 for reducing the pressure from the source; a pressure sensor56 which may also be used as an input into the control system 40; a flowmeter 58 for indicating the flow rate or flow volume; a pressure gauge60 for indicating the pressure in line 10; a pressure relief valve 62;and a one-way valve 64.

Preferably, the two venturi tubes 18, 20, and their controls, aredesigned so as to synchronously feed the same volumes of solutions fromthe two sources 4, 6 even though the viscosities of the two solutionsmay be different. The illustrated system operates at a constantpredetermined water pressure and at a constant ratio of predetermineddilution of the two solutions to the water passing via the branch lines12, 14, through the two venturi tubes 18, 20. Each of these parameterscan be controlled as described above so that the solutions from the twosources 4, 6, are simultaneously and synchronously injected in thedesired predetermined proportions with respect to each other, and alsowith respect to the water flowing through the venturi tubes 18, 20 fromthe source 8.

As indicated earlier, the solution in reservoir 4 is an oxidantsolution, and the solution within reservoir 6 is an amine sourcesolution. Preferably, the latter is a solution of an ammonium salt,preferably ammonium bromide or ammonium chloride or a mixture thereof,most preferably ammonium bromide. The oxidant solution is preferably asolution of calcium hypochlorite or sodium hypochlorite, most preferablysodium hypochlorite. Preferably, the biofilm inhibiting substance isbromide activated chloramine.

Preferably, the biofilm inhibiting substance has a pH of at least 8.5,preferably at least 9.5, just prior to its injection into liquid 3.Preferably the biofilm inhibiting substance is injected at a rate tomaintain in the biofilm inhibiting substance a stable pH of at least8.5.

FIG. 2 illustrates another apparatus, constructed and operative toprovide a biofilm inhibiting substance in accordance with a preferredembodiment of the invention. The apparatus shown in FIG. 2 is similar tothat in FIG. 1, with like numbers denoting elements of the system ofFIG. 2 which are the same as in the system of FIG. 1 and which operatein the same way. The principle difference between the two systems isthat in the system of FIG. 2, the venturi tubes 18, 20 are replaced bypulsatile pumps P₁, P₂. The two pulsatile pumps P₁, P₂ are alsocontrolled by the control system 40 so as to synchronously meter theliquids from the two reservoirs 4, 6, via feed lines 26, 28, in a mannersimilar to that of the venturi tubes 18, 20, in the system describedabove with respect to FIG. 1, except that the liquid pumped out of pumpsP₁ and P₂ is mixed with the water in branch lines 12, 14 at mixers M₁,M₂ as the water in branch lines 12, 14 flows to mixer 21 and then tooutlet line 16. Pulsatile pumps P₁ and P₂ may be replaced by other typesof pumps, such as peristaltic pumps and the like.

The present invention will be better understood through the followingillustrative and non-limitative examples of preferred embodimentsthereof.

Example 1 Formation of Biofilm in a Model System

Formation of biofilm on stainless steel coupons in the presence orabsence of an oxidizing biocide or a biofilm inhibiting substance wasevaluated in the laboratory. The test system consisted of (a) threeclosed flasks each containing 20 L nutrient-rich medium (dilutedthree-fold from its recommended use concentration), (b) three closedcells containing stainless steel coupons hanging freely, and (c) threeidentical circulation pumps, each pump connected via plastic pipes toone of the flasks and to one of the cells. The system was placed in athermostatic room at 35° C.

An innoculum containing a mixed culture of slime-forming bacteria whichhad been isolated from a paper machine was added to each of the flasks.An oxidizer containing a 5 ppm mixture (expressed as total Cl₂) ofbromochlorodimethylhydantoin (an oxidizing biocide which is a source ofHOBr and HOCl) (hereinafter “mixed halogens”) was added to the firstflask once a day for the duration of the trial (4 days). A biofilminhibiting substance, viz. bromide activated chloramine (hereinafter“Fuzzicide BAC”), which can also function as a biocide when applied toplanktonic microorganisms, freshly prepared as described in connectionwith FIG. 1 and in accordance with U.S. Pat. No. 5,976,386 (2.5 ppmexpressed as total Cl₂) was added to the second flask once a day for theduration of the trial. The third flask served as a control for the twoflasks treated with the oxidizing biocide or biofilm inhibitingsubstances. The “Fuzzicide BAC” biocide was produced in a specificfeeding system consisting of two lab pulsatile feeding pumps capable offeeding small volumes (less than 100 μl) per minute with a high pulsefrequency. A diluted solution of sodium hypochlorite in deionized (DI)water (˜8000 ppm as total chlorine) was fed with one pump; a dilutedsolution of ammonium bromide (12500 ppm) was fed with the second pump.The two diluted solutions were synchronously mixed in a short glass pipeto form a pre-injection solution of biofilm inhibiting substance, usinga pH meter to control and check the stability of the biofilm inhibitingsubstance formed. The biofilm inhibiting substance was fed to the testsystem immediately as it was produced. The pre-injection solution ofbiofilm inhibiting substance contained 3500-4000 ppm as total chlorine;the pH was ˜9.5.

On days 2 and 4, each closed cell was opened and 2 coupons wereaseptically removed from each cell. At the same time samples of thecirculating medium were taken as well. Sampling was conducted afterfeeding the daily slug dose of biocide.

Each sample of medium was serially 10-fold diluted in sterile salinesolution and plated in molten agar. Each coupon was thoroughly rinsed toremove any adhered particles, aseptically scraped, and the materialremoved by scraping was quantitatively dispersed in saline solution,vortexed, serially 10-fold diluted and plated in molten agar. Viablecounts of microorganisms were taken after 48 h of incubation at 35° C.Viable counts of cells in the medium are presented as colony units (cfu)per ml; viable counts on the coupon surfaces are presented as cfu/cm².The results are tabulated in Table 1.

After two days the viable counts in the media samples (i.e., planktonicmicroorganisms) were similar in both samples which had been exposed tothe oxidizing biocide or the biofilm inhibiting substance, and theviable counts were only slightly higher in the control sample. Asignificant biofilm was found to be growing on the control coupon after2 days, a smaller but significant microbial population was growing onthe coupons treated with mixed halogens, while the coupons treated withFuzzicide BAC remained clean. After four days, the medium control sampleexhibited a steady count of planktonic microorganisms similar to thecount on day 2, the medium sample treated with mixed halogens exhibitedsome control of planktonic microorganisms (˜10-fold reduction in viablecount), and the medium sample treated with Fuzzicide BAC exhibitedcomplete control of planktonic microorganisms (within detection limits).With respect to the growth on the coupons, after 4 days the coupons ofthe control test exhibited a small increase in the viable count ofbiofilm bacteria compared to the results on day 2, and the couponstreated with mixed halogen exhibited a 3-fold increase in the viablecount of biofilm bacteria compared to day 2. The coupons of the systemtreated with Fuzzicide BAC remained clean.

TABLE 1 Viable Counts Viable Counts After 2 Days After 4 Days Type ofTreatment cfu/ml cfu/cm² cfu/ml Cfu/cm² Mixed Halogens (5 ppm 9 × 10⁶ 27 1 × 10⁶  95 expressed as Cl₂) Fuzzicide BAC (2.5 1 × 10⁶  <27* <100* <27* ppm expressed as Cl₂) Control 1.5 × 10⁷   3645 1.3 × 10⁷   4050*These values represent the lower detection limit of the equipment used,and therefore are expressed as inequalities - it is possible that theviable counts were actually lower than the numbers recited here.

Example 2 Waste Water Fouling Control

Treated wastewater was piped from a wastewater treatment plant to alocation 7 kilometers away. Over the course of years, it was noted thatthe pipes became clogged and the water flow rate through the pipesdecreased. Use of an exceedingly high concentration of Cl₂ (feeding upto 50 ppm, i.e. addition of NaOCl at a level of up to 50 mg/l(calculated as Cl₂)) was found to be ineffective for improving waterconductivity in the pipes. Mechanical cleaning (“pigging”) of the pipesresulted in a significant improvement in water conductivity immediatelyafter cleaning, but this improvement lasted only a few days, after whichtime the pipes attained the level of clogging observed prior to thepigging of the pipes.

Use of the present invention was effective in controlling the biofilm.Prior to beginning a course of treatment using the present invention,the Hazen-Williams coefficient (HW) in the pipe was determined to be˜90. (The Hazen-Williams coefficient is used to express water flowthrough industrial pipes. It is calculated using the formula

${P = \frac{2340 \times B^{1852} \times s}{C^{1852} \times d^{4870}}},$

wherein P is the friction pressure drop expressed in pounds per squareinch per 1000 feet of pipe length, B is the flow rate in barrels perhour, s is the specific gravity of the liquid, C=a friction factor (theHazen-Williams coefficient), and d is the internal diameter of the pipein inches. P and B are measured for a given pipe, s and d are treated asconstants, and C is calculated. The results are presented as theHazen-Williams coefficient. The higher the number, the better the flowthrough the pipe.) Application of 10 ppm of biofilm inhibitingsubstance, viz. bromide activated chloramine produced in accordance withU.S. Pat. No. 5,976,386 (“Fuzzicide BAC”), expressed as total chlorine,once a day for three hours for 6 consecutive days increased the HW valuefrom ˜90 to ˜104. A combination of “pigging” and dosing 10 ppm FuzzicideBAC (expressed as total Cl₂) produced in accordance with U.S. Pat. No.5,976,386 fed once a day for three hours raised the HW value from ˜104to ˜116. Once the pipe had been cleaned in this manner, it was foundthat feeding of 10 ppm (expressed as total chlorine) of Fuzzicide BACproduced in accordance with U.S. Pat. No. 5,976,386 for three hours,once a week, was effective over a period of months to maintain the HWcoefficient at a constant value, i.e. it inhibited further formation ofbiofilm in spite of the high viable counts of microorganisms in thewastewater. The HW coefficient was constant as long as the biofilminhibiting substance as properly formed and fed to the pipe. A decreasein the HW coefficient was noted when the pipe was not treated properly.This was corrected by increasing the frequency of treatment for a fewdays.

The biofilm inhibiting substance in this example was produced asfollows: a feeding system was constructed, containing a first pulsatiledosing pump which was used to feed up to 300 liters/hour sodiumhypochlorite solution (10-15% w/v), and a second pulsatile dosing pumpwhich was used to feed up to 150 liters/hour of ammonium bromide (38%solution w/v). Waste water (up to 10 m³/h) was used to appropriatelydilute both chemicals. An on-line pH meter controlled the productionprocess and the hypochlorite feeding rate to ensure the production of astable biofilm inhibiting substance. The biofilm inhibiting substancewas injected into the treated waste pipe as it was produced.Concentration of the biofilm inhibiting substance stock solution was3000-4000 ppm; the pH was maintained at 9.5-10.

Example 3

Treated wastewater was pumped through several pipes of 10 m length and 4inches inner diameter in a pilot plant. Biofilm had been growingnaturally on the pipe surfaces for several months prior to thecommencement of treatment. Pressure drop through each pipe was monitoredon-line, and average HW coefficients were calculated. During the trial,control pipes were left untreated, and the remaining pipes were treatedwith either (a) the biofilm inhibiting substance Fuzzicide BAC, producedon-site in accordance with the invention of U.S. Pat. No. 5,976,386, 10ppm expressed as total chlorine for three hours three times a week, or(b) a chloramine produced from ammonium chloride which is part of theprior art preceding U.S. Pat. No. 5,976,386 and U.S. Pat. No. 6,132,628,pre-formed as described in the comparative examples of U.S. Pat. No.6,132,628, applied at 10 ppm (expressed as total chlorine) for threehours, three times a week.

The biofilm inhibiting substance in this example was produced asfollows, using a small feeding system was built specifically for thistrial. Up to 4 l/h sodium hypochlorite and up to 2 l/h Fuzzicide BAC inup to 56 l/h water were fed into the treated pipes. The concentration ofthe biofilm inhibiting substance pre-infection solution was ˜3600 ppmand the pH was 9.2-9.6. A major portion of this stock solution wasdiscarded and only a small portion was fed due to the high excess ofbiocide which was formed with this system and the low feed rate. Asshown by the results presented in Table 2 and FIG. 3, proper biofilminhibiting substance formation was critical for the stability andefficacy of the biofilm inhibiting substance—improper preparation led tothe formation of a product which was significantly less efficacious thanFuzzicide BAC. The biofilm inhibiting substance derived from ammoniumchloride was produced in a dosing system that was copied from theFuzzicide BAC feeding system.

Table 2 and FIG. 3 show the difference in HW between the control pipes(untreated) and pipes treated with Fuzzicide BAC.

TABLE 2 Difference in HW Value Between Fuzzicide BAC-Treated Pipe andControl Pipe HW Difference, Fuzzicide Day of Trial BAC - Control  1*10.12  2 11.25  3 11.62  4 13.04  5* 12.35  6 13.76  7 15.13 26^(##)6.11 27* 6.67 28 7.13 29* 8.75 30 8.93 31 8.93 32 9.02 33* 8.89 34 8.9335 9.12 36* 10.31 37 9.96 38 13.97 *= day on which water in pipe wastreated with Fuzzicide BAC. ^(##)= Between days 7 and 26. the biocidewas improperly prepared, rendering it ineffective and resulting in asignificant lowering of the difference between the HW values in the“treated” and untreated pipes.

As can be seen from Table 2, the effect of Fuzzicide BAC on biofilms isnot necessarily apparent on the day of treatment, but is observable forseveral days afterward (in the form of increased HW value in the treatedvs. untreated pipe). The characteristics of the measured HW coefficientshow that control of the biofilm cells is not maintained via killing ofthe embedded cells. This was confirmed by direct enumeration of thebiofilm cells.

Table 3 shows the results of a comparison of the long-term effects oftreatment of biofilm with Fuzzicide BAC vs. treatment with chloramine.On day 1 of this part of the trial, pipes were treated for 3 hours withFuzzicide BAC or chloramine (each at a concentration of 10 ppm,expressed as total chlorine). The difference in HW value between thebiofilm inhibiting substance-fed pipes and the control pipes wasmonitored on-line for the following 13 days. It was expected that afterbiofilm inhibiting substance feeding was ceased, biofilm growth wouldresume in the treated pipes, leading to a decrease in the HW coefficientin these pipes, while the HW coefficient was expected to remain constantin the control (non-treated) pipe. The differences between the HWcoefficients of the treated pipes and control pipe were monitored, andthe results are presented in Table 3 and FIG. 4.

TABLE 3 HW Diff. Fuzzicide BAC - HW Diff. Chloramine - Day of TrialControl Control 1 14.3 13.0 2 13.7 10.8 3 12.8 10.7 4 16.5 15.0 5 16.015.2 6 16.55 15.15 7 15.8 14.6 8 14.3 12.9 9 16.9 13.0 10 15.3 11.5 1116.0 9.5 12 18.1 8.6 13 17.2 7.0 14 15.0 6.2 15 11.9 4.4 16 9.1 2.4 177.3 0.8 18 7.2 0.7

Example 4 Treatment of a Heavily Fouled Paper Machine in Accordance withthe Present Invention

U.S. Pat. No. 5,789,239 describes a composition and process for theavoidance of slime and/or the removal of biofilm in water-bearingsystems. According to the patent, this objective is achieved in that atleast one glycol component and at least one enzyme component from thegroup consisting of carbohydrates, proteases, lipases and glycolproteases are added to the water. The patent presents the results offield trials to demonstrate how the invention disclosed therein can beimplemented and the efficacy of the method disclosed therein. One of theparameters used therein to monitor removal of biofilm is paper quality,which is measured on-line during paper production. The results presentedin U.S. Pat. No. 5,789,239 show that the statistical distribution ofblack spots, light spots and holes monitored in the finished product didnot differ from previous on-line paper quality results achieved withconventional biocidal treatment.

In the present example, a heavily fouled paper machine was treated withthe inventor's Fuzzicide BAC biofilm inhibiting substance, producedon-site using the apparatus described in the inventor's U.S. Pat. No.5,976,386. The biofilm inhibiting substance was added to the papermachine semi-continuously. The paper machine was not boiled out withcaustic prior to commencement of the trial. Rather, the heavy foulingremained present on the machine surfaces at commencement of the trial.

A specifically designed feeding system was built for this trial. A firstpulsatile pump fed up to 30 l/h of sodium hypochlorite; a secondpulsatile pump fed up to 13 l/h of ammonium bromide. Softened water wasused to dilute the chemicals in order to avoid scale formation. TheFuzzicide BAC feeding system was used to dose at three different feedingpoints along the paper machine. The biofilm inhibiting substanceproduction process was controlled by monitoring the pH of the producedbiofilm inhibiting substance and adjusting the mixing of the ingredientsas necessary. The biofilm inhibiting substance pre-injection solutioncontained 3500-4000 ppm expressed as total chlorine, and the product pHwas 9.6-9.8. The biofilm inhibiting substance pre-injection solution wasreproducible and stable for the duration of this trial and during monthsof constant use on this paper machine.

Dark spots, light spots and holes in the finished paper were recordedon-line and are presented in Table 4 and FIG. 5 (the latter of whichshows holes and spots in an average roll of paper, which weighs 20tons). Results are averaged for each type of paper produced (some ofwhich was produced over a period of more than 24 hours).

TABLE 4 Day of H H Trial LS > 20 LS 5-20 DS > 15 DS 5-15 H > 20 10-205-10 1 0 17 0 4 4 10 18 2 0 74 0 1 3 40 65  4* 1 93 4 23 4 19 36 5 1 3681 9 38 143 291 6 0 390 1 31 15 57 363 7 1 950 9 48 148 361 509 8 1 51815 45 69 208 417 9 6 979 16 63 56 156 2266 11* 0 1392 6 36 117 382 1152LS = light spots; DS = dark spots: H = holes; sizes given inmicrometers. *Results on day 4 include data from day 3. Results on day11 include data from day 10.

The steady increase in holes and spots over time from the day oftreatment was due to particles of biofilm, of different sizes andcolors, which broke off from the machine surface with increasingfrequency as a result of the treatment with Fuzzicide BAC.

On the 12^(th) day of the trial, the paper machine was stopped forcleaning. This revealed surfaces covered with a mass of small particlesof biofilm which had broken off from the main area of biofilm growth andhad dispersed in the water of the machine while the machine surfaceswere being cleaned.

Following the cleaning of the paper machine, paper production wasresumed, with addition of the biofilm inhibiting substance Fuzzicide BACto the process water. FIG. 6 shows dark spots, light spots and holesrecording during paper production in this period. In comparison to FIG.5, the total quantity of spots and holes recorded remained relativelysmall throughout this period, indicating that application of the biofilminhibiting substance prevented re-formation of biofilm on the surfacesof the paper machine.

Example 5 Inactivation of Catalase

Laboratory tests were conducted in flasks containing 100 ml of deionized(DI) water and using catalase (Merck, enzyme was diluted in salinesolution to a final concentration of 26 units per ml) and biofilminhibiting substance (Fuzzicide BAG or monochloroamine (MCA)). Freshlyprepared biofilm inhibiting substance was added to the appropriateflasks containing diluted catalase at a pre-defined feed rate. Thecontents of the containers were mixed for 60 minutes at room temperatureprior to addition of H₂O₂ (to a final H₂O₂ concentration of 3.5 g/l).After addition of the H₂O₂, the mixture was allowed to mix for 30minutes at room temperature, at which point H₂O₂ residues were measuredin each flask in accordance with the Dr. Lange Cuvette Test LCW 058,measured with LASA 20 (based on Jander/Blasius. Lehrbach derAnalytischen and Praparative Anorganischen Chemie, as described in theHandbook of Photometrical Operation Analysis (October 1997)). Theresults, which are expressed and presented as total Cl₂, are summarizedin Table 5. Residues of Fuzzicide BAC and MCA were measure with a Hachpocket colorimeter.

TABLE 5 Biofilm inhibiting BIS Concentration Catalase, Initial H₂O₂Residual H₂O₂ substance (BIS) (ppm, as total chlorine) units/mlconcentration, % g/L concentration, % of 3.5% g/l NH₄Br + NaOCl 8.1 263.5 21.4 NH₄Br + NaOCl 60 26 3.5 100 NH₄Br + NaOCl 140 26 3.5 100NH₄Cl + NaOCl 6.7 26 3.5 6.8 NH₄Cl + NaOCl 58 26 3.5 97.1 NH₄Cl + NaOCl128 26 3.5 99.4 NH₄Br + NaOCl 60 0 3.5 100 None 0 26 3.5 ~0 None 0 0 3.5100

These results show (1) that the enzyme was highly active in degradingH₂O₂, (2) that neither chloramine nor Fuzzicide BAC oxidized hydrogenperoxide and (3) that catalase was completely inactivated by chloramineand by Fuzzicide BAC only at a high dosage (˜60 ppm or higher as totalCl₂) which is much higher than the feed level which, as illustrated inthe previous examples, is used to inhibit the biofilm-developingpotential of collections of microorganisms and indirectly bring aboutdisintegration of biofilms. At a dosage level of 10 ppm and lower(expressed as total chlorine), the inventor's biofilm inhibitingsubstances inactivated catalase to an insignificant degree, if at all.

MCA and Fuzzicide BAC were prepared in the lab using procedures similarto those described above for field tests. Sodium hypochlorite wasdiluted in DI water to a final concentration of 6000 ppm expressed astotal chlorine. Ammonium bromide solution (equimolar to 1.1 mole of thediluted sodium hypochlorite solution, 10% excess on a molar basis) andammonium chloride solution (equimolar to 1.1 mole of the dilutedhypochlorite solution, 10% excess on a molar basis) were prepared. Thediluted hypochlorite (50 ml) was added dropwise to 50 ml of theappropriate ammonium salt while the pH was constantly measured. Thebiofilm inhibiting substance concentration in the produced stocksolution was immediately measured and the biofilm inhibiting substanceat the appropriate feed level was immediately added to the test flasks.

For all practical purposes, MCA and Fuzzicide BAC are ineffective indeactivating peroxide-degrading enzymes when administered at a feed ratelevel optimized for inhibiting biofilm development at reasonable cost.Thus the mode of action of these biofilm inhibiting substances againstthe peroxide-degrading enzyme catalase must operate according to amechanism other than direct inactivation at the enzymes. The presentexample shows that unlike HOCl and HOBr, which readily react with H₂O₂,MCA and Fuzzicide BAC do not oxidize H₂O₂. This property enables MCA andFuzzicide BAC to be used as biofilm inhibiting substances in thepresence of high background concentrations of H₂O₂ or in mixturescontaining H₂O₂. Unlike oxidizing biocides which have been used in theart to prevent biofilm growth by killing microorganisms embedded in thebiofilm. MCA and in an especially preferred embodiment of the presentinvention Fuzzicide BAC may be used in the presence of or in combinationwith other enzymes which may, for various purposes, be added to aprocess medium, especially an aqueous process medium.

Example 6 Field Trial at a De-Inking Plant

A de-inking system had been using 7-10 kg H₂O₂ per ton of waste paper.Previous attempts to control the enzymatic degradation of H₂O₂ usingconventional biocides like glutaraldehyde did not yield cost-effectiveresults on this system. A parallel de-inking system at the same plant,utilizing a similar de-inking process on waste paper front the samesource, was successfully treated with a commercial chemical formulationcontaining glutaraldehyde: the average H₂O₂ consumption rate in thisdeinking process was reduced to ˜4 kg H₂O₂/ton of waste paper.Measurements conducted prior to the commencement of the trial with theFuzzicide BAC technology showed that a high microbial load was presentin various parts of the deinking plant, indicating a build-up of heavyslime. Despite the high initial dosage of H₂O₂, negligible residues ofH₂O₂ were found at various points along the system's flow path.

Fuzzicide BAC, produced on-site with a production feed system asdescribed in U.S. Pat. No. 5,976,386, was then fed continuously into theprocess water for a period of 850 minutes. The biofilm inhibitingsubstance was produced on-site in a specifically designed dosing systemsimilar to the dosing system described in Example 4. The reaction pH wasmaintained at 9.8-10.0. The production process was controlled to ensuresynchronous metering of the two chemicals, continuous mixing at thepredetermined molar ratio and reproducible production of a stablebiofilm inhibiting substance stock solution for the duration of thetrial and longer. The initial Fuzzicide BAC dosing rate was 170 g/tonexpressed as total Cl₂. After 850 minutes the dosing rate was reduced to85 g/ton expressed as total Cl₂ by feeding the biofilm inhibitingsubstance semi-continuously. Various parameters were monitored duringstart-up of the trial: Residual biofilm inhibiting substance wasmeasured (using a Hach pocket colorimeter, total Cl₂, based on the DPDmethod adapted from Standard Methods for Examination of Waste and WasteWater). Residual hydrogen peroxide was measured using either LASA 20with the LCW 085 method, based on the method of Jander/Blasius, Lehrbuchder Analytischen and Praparative Anorganischen Chemie, as described inthe Handbook of Photometrical Operation Analysis by Dr. Lange for theLASA 20, October 1997 (in cases of high concentration), or Merck TestStrips (0.5-25 ppm). When necessary, samples were diluted with DI water.

The activity of H₂O₂-degrading enzymes in the process water was measuredaccording to the following procedure: a commercial solution of H₂O₂ wasdiluted with DI water to a final concentration of 100 g/l water (10%).One ml of the diluted H₂O₂ solution was added to 9 ml of a sample takenfrom the treated de-inking process water to form a final feed rate of 10g/l H₂O₂. The combined sample was incubated at room temperature for 15minutes, at which time residual H₂O₂ was measured. Hydrogen peroxidediluted in DI water served as a control. The residual concentration ofH₂O₂ was low when the enzymes effectively degraded H₂O₂, whereas theresidual concentration of H₂O₂ was high and close to the H₂O₂ feed rateas the H₂O₂-degrading enzymes became less effective or as theconcentration of enzymes in the process water decreased. The results as% of the H₂O₂ remaining the process water after the defined contact timeare presented in Table 6. Adenosine triphosphate (ATP) measurements inTable 6 are based on the following process: during the change from ATPto Adenosine monophosphate (AMP) in the presence of luciferin andluciferase, a defined quantity of light is emitted per ATP molecule.This emitted light is measured by a photometer. The results are given inrelative terms and are thus relative and snot absolute (RLU=relativelight unit). The values can be correlated with microbial activity in thesense that for high viable counts, a high ATP measurement is obtained,and vice versa.

TABLE 6 Reduction in Catalase Residual Activity, as % Fuzzicide BAC,Time, of initial H₂O₂ ATP Residual ppm as total min. concentration (RLU)H₂O₂, ppm chlorine 0 37.6 132276 0 0 100 17.8 6340 ~5 0.7 240 54.7 2861~5 1.45 850 92 535 >25 1.4 1500 135.1 3568 >250 0.7

The sharp decrease in ATP following commencement of the trialdemonstrates effective control of planktonic microorganisms (free livingcells) in the pulper. As expected on the basis of the inventor's earlieraforementioned U.S. patents, the level of ATP continued to decreasethroughout the period of continuous dosing, even though the measuredresidues of Fuzzicide BAC were not exceedingly high. The apparentincrease in catalase activity between 0 and 100 minutes is due todegradation of the biofilm and consequent release of material from thebiofilm, including microorganisms, catalase and other peroxide-degradingenzymes into the process water.

After 850 minutes, when measurable residues of H₂O₂ were detected insamples taken from the pulper, the dosing regime was changed: continuousfeed was replaced by semi-continuous feed and the total feed rate wasreduced to 50% of its initial value, to 85 g (expressed as total Cl₂)per ton of pulp. As expected, the ATP value increased, reflecting anincrease in the count of planktonic microorganisms, with a decrease inboth feed rate and residue of total Cl₂.

In spite of the increase in ATP and in viable counts, H₂O₂-degradingenzyme activity decreased as the treatment progressed, and wasaccompanied by an increase in the concentration of available H₂O₂measured in the process water. After 1500 minutes, H₂O₂-degrading enzymeactivity appeared to be wiped out, even though the biocide feed rate wasdecreased at 850 minutes, and ATP concentrations increased between 850and 1500 minutes.

After about 48 hours of semi-continuous dosing of the biocide, the feedrate of H₂O₂ needed in order to maintain bleaching set point was reducedto ˜4 kg/ton. After a few more days, it was found that the H₂O₂ feedrate could be further reduced to ˜2.2 kg/ton and yet the definedde-inking bleaching targets could be maintained at this reduced feedrate.

Example 7 Fuzzicide BAC Efficacy and Viable Counts

During a field trial with Fuzzicide BAC in a paper machine used toproduce printing and typing paper, viable counts of microorganisms,principally bacteria, were monitored in the white-water silo (ww) and inthe machine chest (Mchest). Process water samples were drawn andimmediately inactivated with sodium thiosulfate to degrade any residueof the biofilm inhibiting substance. Samples were then serially ten-folddiluted in a Trypton (DIFCO) saline dilution medium. The diluted sampleswere plated in molten R2A Agar (hereinafter=“total count”) and in moltenPlate Count agar containing a high excess of glucose (hereinafter “slimeformers”). The agar solidified at room temperature and the plates wereincubated at 35° C. for 48 h. Viable cells were counted and the resultsare presented in Table 7 below and in FIG. 7. Two different treatmentperiods were noted: the biofouling-cleansng period, during whichtreatment the biofilm inhibiting substance brought about disintegrationof the existing biofilm (see also Example 4), and the normal operationperiod following the cleaning period, when the paper machine operatednormally and application of the biofilm inhibiting substance was used tomaintain smooth operation of the paper machine (compare to FIG. 6).

Table 7 and FIG. 7 show that during the initial cleaning period, theviable counts in process water samples taken from the silo contained10³-10⁴ viable cells per ml, irrespective of whether the residue of theFuzzicide BAC biofilm inhibiting substance was present in high or lowconcentration. Almost all of the silo samples contained a significantnumber of colonies, which grew on a high glucose medium. A similarphenomenon was observed in samples taken from the Mchest (results notshown), which exhibited even higher numbers of both total counts andcells which grow in the presence of high glucose content.

TABLE 7 Silo/residual Cl₂ silo/slime formers silo/total count Day ofTrial (ppm) (cfu) (cfu) 1 5.85 1.0 × 10¹   1 × 10⁰ 2 6.3 5.92 × 10³ 5.68 × 10⁴  3 1.98 2.0 × 10² 4.8 × 10⁴ 4 2.64 1.0 × 10⁰ 7.6 × 10³ 6 2.181.2 × 10² 3.8 × 10³ 7 3.2 1.0 × 10⁰ 4.0 × 10³ 8 4 4.0 × 10¹ 1.68 × 10³ 9 5.05 2.2 × 10² 5.0 × 10³ 10 5.1 1.0 × 10⁰ 1.18 × 10³  13 2.72 1.0 ×10¹ 2.4 × 10³

As shown in Table 8, once the paper machine was clean, a significantreduction in total count was found in the water samples.

TABLE 8 silo/residual Cl₂ silo/slime formers silo/total count Day ofTrial (ppm) (cfu) (cfu) 16 2.94 1.0 × 10⁰ 2.0 × 10² 17 3.08 1.0 × 10⁰6.0 × 10¹ 20 2.56 1.0 × 10⁰ 3.0 × 10² 21 2.26 1.0 × 10⁰ 7.5 × 10² 24 2.21.0 × 10⁰ 1.0 × 10² 27 3.62 1.0 × 10⁰ 1.1 × 10²

Taken together, these results indicate that (a) as long as the papermachine was heavily fouled, many if not most of the viable cells,including those embedded in the biofilm, readily grew on a medium havinga high glucose content, indicating the presence of enzymes capable ofefficiently and quickly degrading glucose, whereas (b) in a cleanmachine treated with Fuzzicide BAC, the viable cells were unable to growon a glucose-rich medium, indicating that these cells did not containenzymes capable of efficiently and quickly degrading glucose at a highconcentration, irrespective of whether total counts of viable cells onR2A medium were high or low. These results can be compared with FIGS. 3and 4, which also show that treatment with biofilm inhibiting substanceaccording to the present invention brings about the disintegration ofbiofilm in biofouled machines and prevents the re-formation of biofilmin clean machines.

Example 8 Effect of Fuzzicide BAC on Papermaking Efficiency

In a papermaking machine, Fuzzicide BAC was fed intermittently intovarious parts of the machine. Quick loss of residual Fuzzicide BAC inthe machine was observed, the main loss in residual Fuzzicide BAC takingplace in the pulpers, specifically in the dry broke pulper. (The drybroke pulper receives paper produced by the machine but which is ofunacceptable quality for shipment to customers; this paper is re-used inthe paper making machine). It was observed that in the pulpers, the lossin residual biocide was accompanied by a sharp increase in ATP. Initialinvestigations suggested that the observations were attributable tosub-optimal disinfection in the size press, where starch used to coatthe paper is present and provides a good medium to support the growth ofmicroorganisms.

At the same time the loss of residual Fuzzicide BAC and increase in ATPin the pulper was observed, a sharp increase in ATP in the Machine Chestand Head Box, as well as in the clear water, was also observed.

Although the ATP in the pulpers was high, the results in the WhiteWater, which is machine recycled water were still within acceptableparameters.

In order to determine if the loss of residual Fuzzicide BAC was due toproblems in the wet end chemistry, the amount of cationic starch beingfed to the Machine Chest was reduced by 50%, and 11 hours later thedosage of polyaluminium chloride (PAC), a floculant to aid inagglomeration of fibers and particles in the headbox, was increased by20%. Dry broke was still used during this period. The effect on totalcalcium carbonate retention and precipitated calcium carbonate (PCC)(ash) retention were similar. Changes in feed rate of cationic starchand PAC did not affect the retention significantly.

Five hours after the amount of cationic starch being fed to the MachineChest was reduced, the dosing rate of Fuzzicide BAC was increased by65%. A sharp drop in the concentration of suspended material and PCC inthe White Water was noted two hours thereafter, followed by a steadyimprovement in retention during the following 17 hours. The improvementin retention paralleled a steady, slow increase in residual chlorine.

It will be appreciated by persons skilled in the art that the presentinvention is not limited be what has been particularly shown anddescribed hereinabove. Rather the scope of the present inventionincludes both combinations and subcombinations of the features describedhereinabove as well as modifications and variations thereof which wouldoccur to a person of skill in the art upon reading the foregoingdescription and which are not the prior art.

1-241. (canceled)
 242. A method for reducing effects of an enzymeproduced by a collection of microorganisms attached to a surface in anindustrial water environment, the method comprising: causing a substanceformed by the reaction of a hypochlorite oxidant and an amine source tocontact said collection of microorganisms when attached to said surfacein said industrial water environment by intermittently feeding saidsubstance into water in said industrial water environment, wherein: saidindustrial water environment includes planktonic microorganisms, duringeach feeding of said intermittently feeding said substance, saidsubstance is fed for a duration and at a concentration sufficient tosubstantially eliminate the production of said enzyme by said collectionof microorganisms until the next feeding of said intermittently feedingsaid substance, but insufficient to completely eradicate said collectionof microorganisms and insufficient to inactivate said enzyme, and viablecounts of said planktonic microorganisms increase and activity of saidenzyme decreases in said water during said intermittently feeding saidsubstance.
 243. A method according to claim 242, wherein said substanceis introduced into said water in said industrial water environmentperiodically with a duty cycle of less than 1:2.
 244. A method accordingto claim 242, wherein the duration between each feeding and said nextfeeding duration is between about 5 minutes and about 4 hours.
 245. Amethod according to claim 242 and wherein said collection ofmicroorganisms is attached to a durable surface.
 246. A method accordingto claim 242 wherein said first duration is about 3 hours.
 247. A methodaccording to claim 242 wherein said first duration is about 5 minutes.248. A method according to claim 242 wherein said collection ofmicroorganisms is attached to a consumable surface.
 249. A methodaccording to claim 242, wherein said amine source is selected fromammonium bromide and ammonium chloride.
 250. A method according to claim242, wherein said introducing said substance includes generating saidsubstance in real time.
 251. A method according to claim 250, whereinsaid generating said substance in real time includes producing apredetermined dilution of a hypochlorite oxidant, producing apredetermined dilution of an amine source, synchronously metering thetwo dilutions into a mixer to continuously mix therein according to apredetermined ratio to produce the substance having an effective amountof reproducibility, stability and efficacy in situ in the mixer.
 252. Amethod according to claim 250, wherein said generating said substance inreal time includes continuously and synchronously injecting a quantityof hypochlorite into a first stream of water passing through a firstconduit to produce therein a predetermined dilution of the hypochlorite,continuously and synchronously injecting a quantity of an amine sourceinto a second stream of water passing through a second conduit toproduce therein a predetermined dilution of the amine source andcontinuously and synchronously injecting the first and second streamsinto a mixer according to a predetermined ratio to produce said biofilminhibiting substance in situ in the mixer.
 253. A method according toclaim 242, wherein said substance includes an effective amount ofbromide activated chloramine.
 254. A method according claim 242 whereinsaid substance has a pH of at least 8.5 before being applied to saidwater.
 255. A method according to claim 242 wherein said water has a pHof between about 5 and about 10.5 before said substance is applied tosaid water.
 256. A method according to claim 242, wherein said substanceis applied to said water to a concentration of 0.5-300 ppm expressed aschlorine.
 257. A method according to claim 242, wherein said substanceis applied to said water to a concentration of 3-10 ppm expressed aschlorine.
 258. A method according to claim 242, wherein said aminesource has a concentration of about 0.1 wt. % to about 50 wt. %.
 259. Amethod according to claim 242, wherein said hypochlorite oxidant isselected from the group consisting of sodium hypochlorite and calciumhypochlorite.
 260. A method according to claim 242, wherein a base issynchronously added to said amine source to stabilize the biofilminhibiting substance.
 261. A method according to claim 242, wherein saidoxidant has a concentration of between 0.1 wt. % and 15 wt. % expressedas Cl2
 262. A method according to claim 242, wherein said enzyme is ahydrogen peroxide-degrading enzyme (HPDE) or a starch-degrading enzyme.263. A method according to claim 262, wherein said enzyme is a hydrogenperoxide-degrading enzyme and said collection of microorganisms ispresent at an interface between a surface and de-inking or bleachingprocess water, and the persistence of hydrogen peroxide in saidde-inking or bleaching process water is increased by application of saidbiofilm inhibiting substance.